Caged membrane-permeant inositol phosphates

ABSTRACT

Caged acyloxyalkyl esters of phosphate-containing inositol phosphates which are capable of permeating cell membranes. The second messengers are protected (caged) at the 6-hydroxyl, with a photolabile group. Once inside the cell, the ester derivatives undergo enzymatic conversion to remove the acyloxyalkyl ester groups. The resulting caged compound remains biologically inactive until exposed to ultraviolet (UV) light. Upon UV light exposure, the active form of the second messenger is released within the cell.

This invention was made with Government Support under Grant No.NS-27177, awarded by the National Institute of Health. The Governmenthas certain rights in this invention.

This is a continuation-in-part of application Ser. No. 08/475,758, filedJun. 7, 1995 which is a continuation in part of application Ser. No.08/045,585, filed April 9, 1993, now U.S. Pat. No. 5,693,521.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to biologically importantphosphates such as second messengers. More particularly, the presentinvention relates to modifying second messengers to form derivativeswhich can be introduced into a cell without disrupting the cellmembrane. Once inside the cell, the derivative is converted back to thebiologically active second messenger.

2. Description of Related Art

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Forconvenience, the reference materials are numerically reference andgrouped in the appended bibliography.

Second messengers are ions or small molecules that carry informationfrom the cell membrane to targets on the inside of the cell. They play amajor role in biological signal transduction and amplification (1). Acommon feature of most of the known second messengers, such as adenosine3',5'-cyclic monophosphate (cAMP) (2,3), guanosine 3',5'-cyclicmonophosphate (4) (cGMP), myo-inositol-1,4,5-triphosphate (1,4,5)IP₃) ormyo-inositol-1,3,4,5-tetrakisphosphate (1,3,4,5)IP₄) (5), is thepresence of phosphates. The correct number and position of thesephosphates is essential for biological specificity and also confersextreme hydrophilicity (6,7).

The hydrophilicity of second messengers prevents endogenously generatedmolecules from leaking out of cells. As a result, high sensitivity ismaintained within the responding cell and freedom from cross-talkbetween neighboring cells is achieved. However, the membraneimpermeability of second messengers also makes deliberate extracellularapplication difficult or ineffective (2,6,8), even though suchintervention would often be very useful for research or therapeuticreasons.

One approach for introducing phosphate containing compounds into cellsinvolves using protective groups to reversibly convert the negativelycharged phosphate compound into a neutral compound for transport throughthe cell membrane. The protective group is chosen so that it isenzymatically cleaved from the phosphate compound inside the cell toproduce the original phosphate compound. For example, lipophilic,intracellularly hydrolyzable derivatives have been useful for amino,hydroxyl, and carboxylate moieties (9-12). Acetoxymethyl (AM) esters ofpolycarboxylate cation indicators and chelators are have also been used(12-14). Analogous acyloxyalkyl esters applied to phosphates are known,but they have been less widely exploited (15).

On simple model phosphates, uses of AM esters have been limited topotential therapeutic drugs, such as phosphonoformate (foscarnet) (16),antiviral nucleotides such as 5-fluoro-2'-deoxyuridine monophosphate(17,18), and a 3-phosphonate-containing inhibitor of the insulinreceptor kinase (19). The phosphonoformate esters proved not to bebiologically useful due to failure to hydrolyze to the correct products(16), but esterification was found to enhance the effectiveness of theantiviral nucleotides and kinase inhibitor (17-19).

Considerable work has been done on o-nitrobenzyl esters as photolyzable("caged") derivatives of ATP (20), cyclic nucleotides (21,22), andinositol phosphates (23). However, the emphasis has been on producing akinetically fast and complete transition from a monoester to the activefreed phosphate metabolite (24,25), rather than as a general means ofachieving membrane permeability. In addition, nitrobenzyl esters becomecumbersome if more than one are required to mask negative charges,because multiple groups add considerable bulk and require high doses ofUV to cause cleavage of all the groups.

Although numerous different myo-inositol polyphosphates are possible,only about a dozen have been found in cells. Their intracellularfunctions are controversial or unknown, except for myo-inositol1,4,5-triphosphate (IP₃), whose role as an intracellular secondmessenger to release Ca²⁺ from internal stores is unquestioned (26). Thenext most studied inositol polyphosphate is myo-inositol1,3,4,5-tetrakisphosphate (IP₄), which is believed to cooperate with IP₃to open Ca²⁺ -channels in the plasma membrane (27-30) or to resequesterCa²⁺ released by IP₃ (31, 32). However, these hypotheses remaincontroversial (33-37).

Almost nothing is known about intracellular functions for other inositolpolyphosphates. Detailed dissection of the roles of inositolpolyphosphates is often difficult if they are endogenously generated inresponse to agonists, since such stimulation may affect multiplereceptors, G proteins, diacylglycerol formation, multiple inositolpolyphosphates, and yet other transduction pathways. Direct introductionof specific inositol polyphosphates is often preferable.

The high negative charge of inositol polyphosphates results innegligible passive permeability through membranes. Existing methods forintroducing inositol phosphates include microinjection, patch-clamptechniques, and permeabilization by electroporation, detergents likesaponin, or removal of extracellular Ca²⁺. All these methods breach theplasma membrane and jeopardize the more complex functions and long-termviability of the cells. Furthermore, microinjection and patch techniquescan only be applied to a few cells at a time.

In view of the above, there is a need to provide new compounds andprocedures for increasing the membrane permeability of second messengersso that they can be introduced effectively into cells in amounts whichare useful for investigational or therapeutic purposes.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided for increasing the permeability of phosphate-containing secondmessengers into a cell without disrupting the cell membrane. One aspectof the invention involves esterifying the phosphate groups present inthe second messenger molecule to form an acyloxyalkyl ester derivative.The acyloxyalkyl ester of the second messenger has a neutral charge andtherefor can permeate into the cell without disrupting the cellmembrane. Once inside the cell, the esters are cleaved to convert themolecule back to its biologically active form. Acetoxymethyl estersworked well for cyclic nucleotide esters but proved insufficientlyhydrophobic for inositol phosphates. Propionyloxymethyl andbutyryloxymethyl esters are preferred for the latter class.

We discovered that esterification of the inositol phosphates withacetoxymethyl groups did not provide a sufficient increase in cellpermeability to be useful. We discovered that more hydrophobic esterswere required. Replacement of acetoxymethyl groups withpropionyloxymethyl and butyryloxymethyl groups produces inositolphosphate esters which have much greater cell permeability rates whilestill being amenable to intracellular cleavage to form the activeinositol phosphate. Inositol phosphates which have this high degree ofcell permeability include those having the formula ##STR1## wherein A₁to A₆ is H, OH, F or ##STR2## wherein R is an alkyl group having from 2to 6 carbon atoms and R' is H or CH₃ or R is CH₃ and R' is CH₃ andwherein at least one of A₁ to A₆ is a phosphoester having the formulaset forth above.

As a feature of the present invention, it was discovered that therelease of active inositol polyphosphates within the cell can beaccurately controlled by protecting or caging the 6-hydroxyl group ofthe inositol polyphosphate ester with a photolabile protecting group.Upon entry of the caged inositol polyphosphate ester into the cell, allof the protecting groups, except the caging group protecting the6-hydroxyl group, are enzymatically removed. The resulting cagedinositol polyphosphate remains in the cell in a inactive form until itis uncaged by exposure to ultraviolet light. This photoactivationfeature allows one to accurately control the time when the activeinositol polyphosphate is released within the cell.

The caged membrane-permeant second messengers include compounds whichhave the following formulas: ##STR3## Wherein R₁ is H, --CHO, COOCH₃ or--COCH₃ ; R₂ is H or --P(O)(OR₄)(XR₄);.or R₁ and R₂ together are --CH₂--, --CHMe--, --CMe₂ --, --CH(OMe)--, --CMe(OMe)-- or --C(OMe)₂ --; R₃is a photolabile protecting group;

R₄ is ##STR4##

; and

X is O or S.

The above described and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing description when taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is four graphic representations of the results of tests showingthe kinase activation capability of Bt₂ cAMP/AM in comparison to otherderivatives.

FIG. 2A is a graphic representation of the results of tests showing theinitiation of CI⁻ secretion by Bt₂ cAMP/AM.

FIG. 2B is a graph showing a comparison of chloride secretion initiatedby Bt₂ cAMP/AM and other derivatives.

FIG. 3A shows the results of tests demonstrating the ability of Bt₂cAMP/AM to disperse fish dermal chromatophores.

FIG. 3B is a graph showing test results demonstrating the reversibilityof the dispersion shown in FIG. 3A.

FIG. 4 depicts the synthesis scheme preparing 3',5'-cyclic monophosphateacetoxymethyl ester.

FIG. 5 is a schematic representation of the synthesis of exemplaryesters of inositol phosphates.

FIG. 6 depicts test results showing permeation of inositol triphosphatepropionyloxymethyl ester (IP₃ /PM) into REF-52 fibroblast cells.

FIGS. 7 depicts test results showing permeation of IP3/PM intoastrocytoma cells at a dose of 20 μm in a medium containingextracellular Ca²⁺.

FIG. 8 depicts test results showing permeation of IP₃ /PM at a dose of60 μm into astrocytoma cells in a medium lacking extracellular Ca₂₊.

FIG. 9 depicts test results showing permeation of inositol triphosphatebutyryloxymethyl ester (IP₃ /BM) into astrocytoma cells at a dose of 2μm in a medium containing extracellular Ca²⁺.

FIG. 10 is a schematic representation of the synthesis of exemplarycaged esters of inositol phosphates.

FIG. 11 shows exemplary preferred caged esters of inositol phosphates.

FIG. 12 shows details of the first portion of the synthesis of anexemplary caged inositol phosphate. The reference letters in the drawingrefer to reagents used in each step as follows:

a. (1) Bu₂ SnO, toluene, Dean-Stark apparatus, reflux, (2) DMNB-Br,CsF/DMF; b. S-(-)-camphanic acid chloride, TEA, DMAP; c.2-mercaptoethanol, BF₃ /Et₂ O; d. BzCI, pyridine; e. 2-meraptoethanol,BF₃ /Et₂ O

FIG. 13 shows details of the second portion of the synthesis of anexemplary caged inositol phosphate. The reference letters in the drawingrefer to reagents used in each step as follows:

a. K₂ CO₃, MeOH/CH₂ /CI₂ ; b. BzCI, pyridine, DMAP; c.2-mercaptoethanol, BF ₃ /Et₂ O; d. BzCI, pyridine, DMAP; e.2-mercaptoethanol, BF ₃ /Et₂ O; f. Trimethylorthoformate, BF ₃ /Et₂ O,DMF; g. K₂ CO₃, MeOH/CH₂ CI₂ ; h. (1) (CNCH₂ CH₂ O)₂ PN(i-Pr)₂,tetrazole (2) t-BuOOH, CH₂ CI₂ ; i. NH₄ OH, MeOH; j. PM-Br, DIEA/CH₃ CN

FIG. 14 shows the results of tests demonstrating IP₃ -CPM (V-1) inducedCa²⁺ !_(i) increase upon uncaging. (a) Extracellular application of IP₃-CPM (A, 20 μM) had no effect on Ca²⁺ !_(i) even after incubation forabout 5 minutes. Exposure of the cells to UV light (B, 360 nm, 10 s)caused an almost saturating Ca²⁺ !_(i) increase. The Ca²⁺ !_(i) levelremained elevated above the resting level due to the Ca²⁺ influx.Carbachol (C, 200 μM) and thapsigargin (D, 100 nM) gave normal response.Ionomycin (E, 5 μM) was added at the end of the experiment and gave ahuge Ca²⁺ !_(i) increase. (b) Same experiment as (a) except done in Ca²⁺free medium (DPBS, O Ca²⁺, 0.5 mM EGTA). UV exposure of the cellstreated with IP₃ -CPM still gave the same response, showing that theCa²⁺ !_(i) increase resulted from the release of Ca²⁺ from internalstores. The Ca²⁺ !_(i) level soon went back to the resting level becausethere was no extracellular Ca²⁺ with which to maintain a Ca²⁺ !_(i)elevation. Ionomycin was added at the end of the experiment and gave anadditional small Ca²⁺ !_(i) increase, probably from IP₃ insensitivestores. The cells were loaded with fluo-3/AM. During the experiment thecells were bathed in HBS buffered with 20 mM Hepes at pH 7.4 andsupplemented with 2 g/L glucose.

FIG. 15 shows the results of tests demonstrating IP₃ -CPM (V-1) inducedCa²⁺ !_(i) increase upon uncaging in P388D₁ macrophage-like cells.Extracellular application IP₃ -CPM (A, 20 μM) had no effect on Ca²⁺!_(i) even after incubation for about 5 minutes. Exposure of the cellsto UV light (B, 360 nm, 10 s) caused a big Ca²⁺ !_(i) increase. The Ca²⁺!_(i) level went back to the basal level in a short time, indicatingthere was little Ca²⁺ influx. PAF (C, 100 nM) stimulation gaveattenuated response, possibly because the internal stores were notrefilled yet. Ionomycin (D, 5 μM) was added at the end of the experimentand gave a huge Ca²⁺ !_(i) increase. The cells were loaded withfluo-3/AM. During the experiment the cells were bathed in HBS bufferedwith 20 mM Hepes at pH 7.4 and supplemented with 2 g/L glucose.

FIGS. 16-18 are diagrams of exemplary synthesis procedures for makingcompounds in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to modifying second messengers andderivatives thereof which contain phosphates so that they can be easilyintroduced into a cell without disrupting the cell membrane. Theinvention involves esterifying the phosphate groups present in thesecond messenger to form a neutral acyloxyalkyl derivative which canreadily diffuse through the cell membrane. The acyl group may contain upto 6 carbon atoms and is located at the 1 position of the alkyl group.The alkyl group may contain up to 7 carbon atoms. It was found for cAMPand cGMP that acetoxymethyl esters provide optimum cell membranetransport while still being amendable to cleavage from the secondmessenger after entry into the cell. Acetoxymethyl esters of inositolphosphates were found not to be optimally cell permeable. For secondmessengers having multiple phosphate groups, it is preferred that all ofphosphate groups be masked as propionyloxymethyl or butyryloxymethylesters.

The present invention is applicable to increasing the cell permeabilityof a wide variety of phosphate-containing second messengers andderivatives thereof. Preferred exemplary phosphate-containing secondmessengers include cAMP, cGMP, 1,4,5 IP₃, 1,3,4,5 IP₄ and myo-inositol3,4,5,6-tetrakisphosphate. The present invention is also applicable toderivatives of cyclic nucleotides. Exemplary derivatives include the8-substituted derivatives of cAMP or cGMP. The 8-substituted derivativesinclude 8-bromo-cAMP or cGMP, 8-chloro-cAMP or cGMP and8-para-chlorophenylthio (cCPT) cAMP or cGMP.

It is preferred for cAMP and cGMP that the hydroxyl groups of thephosphate-containing second messengers be masked or protected duringsynthesis. Masking with acyl groups having up to 4 carbon atoms ispreferred. Larger acyl groups are not preferred because they are moredifficult for the cell to cleave from the second messenger.

Examples of practice showing the synthesis and use of cAMP/cGMPacetoxymethyl esters in accordance with the present invention are asfollows:

Proton and ³¹ P NMR spectra were obtained in CDCI₃, with residual CHCI₃(δ=7.26), being used as the internal standard for ¹ H spectra. 85%phosphoric acid was used as an external standard for ³¹ P spectra. AllNMR spectra were recorded on either a Varian Gemini-200 (200 MHz) or aGeneral Electric QE-300 (300 MHz) spectrometer and are reported with thefollowing abbreviations: s, singlet; d, doublet; t, triplet; dd, doubletof doublets; m, complex multiplet. Fast atom bombardment massspectroscopy (with glycerol as matrix) and precise mass determinationswere performed by the mass spectroscopy facility of the University ofCalifornia, Riverside. Capillary electrophoresis was performed on aDionex CES.

Pyridine and acetonitrile used in the synthesis were stored overactivated molecular sieve (3 Å) for at least 3 d. All other solventswere purchased in highest purity available and were used as received.N,N-Diisopropylethylamine (DIEA) was distilled from CaH₂. Acetoxymethylbromide (AM-Br) was prepared according to known procedures (38). Allnucleotides were from Sigma. Phenylphosphonic acid was from Fluka,Switzerland. 4-Methylumbelliferylphosphate was from Boehringer, FRG. Allother reagents were from Aldrich.

                                      TABLE 1    __________________________________________________________________________    Structures of Acetoxymethyl Esters of Various Organic Phosphates    comp.         ##STR5##              counter.sup.a ion M*                                    yield.sup.b                                       .sup.31 P-NMR  ppm!    __________________________________________________________________________         ##STR6##              Ag*  72%                                       -9.1    2         ##STR7##              Ag*  98%                                       -2.25    3         ##STR8##              HDIEA*                                    86%                                       18.70    4a/4b 5a/5b         ##STR9##              HDIEA* Ag* HDIEA*                                    59% 52% 40%                                       -8.0/.sup.c -5.0 -5.5/.sup.c -8.5    __________________________________________________________________________     .sup.a M.sup.+  specifies the counter ion for the phosphatecontaining     starting material; HDIEA.sup.+  = diisopropylethylammonium.     .sup.b Yield by weight unless otherwise noted.     .sup.c Shift values for both diastereomers.

The compounds shown in Table 1 were synthesized as set forth below:Compounds 1-3 were synthesized for comparative purposes. Compounds 4a/b(cAMP/AM) and 5a/b (cGMP/AM) are preferred exemplary compounds inaccordance with the present invention.

Synthesis of 4-Methylumbelliferyl Phosphate Bis(acetoxymethyl)ester(1)--The dilithium salt of 4-methylumbelliferyl phosphate (200 mg, 0.74mmol) was dissolved in water and a concentrated solution of silveracetate was added. The disilver 4-methylumbelliferyl phosphateprecipitated immediately and was filtered, washed with water and driedto a shining silver-white powder (yield: 277 mg, 79%). The silver salt(60 mg, 0.13 mmol) was suspended in 1 mL dry CH₃ CN and 50 mg (0.33mmol) AM-Br was added. At frequent intervals, the mixture was treatedfor 2 minutes at a time in an ultrasonic bath (Branson B-220). Frequentmonitoring by ¹ H NMR showed the reaction to be complete after 4 hours.The supernatant was evaporated to dryness to yield 38 mg (72%) of4-methylumbelliferyl phosphate bis(acetoxymethyl)ester (1); ¹ H NMR(CDCI₃, 200 MHz) δ 2.12 (s, 6H),;2.43 (s, 3H), 5.73 (dAB, 4H, J_(AB)=5.5 Hz, J_(PH) =14.2 Hz, --CH₂ --), 6.27 (s, 1H, H3), 7.17-7.25 (m, 2H,H6,H8), 7.59 (m, 1H, H5); ³¹ P NMR (CDCI₃, 121.5 MHz) δ-9.1; MS m/z(M+H)⁺ calcd 401.0638, obsd 401.0625.

Synthesis of Phosphate Tris(acetoxymethyl)ester (2)--Silver phosphate(30 mg, 71 μmol) was suspended in 0.5 mL dry CH₃ CN and AM-Br (22 mg,145 μmol) was added. After frequent sonication for 20 hours at roomtemperature, another 15 mg (100 μmol) AM-Br was added. When thesuspended solid had lost its yellow color, the mixture was centrifuged(1000 rpm, 1 min), and the supernatant was evaporated to dryness and theresidue was washed with dry toluene to give phosphatetris(acetoxy-methyl)ester (2) as a clear oil (yield 98%); ¹ H NMR(CDCI₃, 200 MHz) δ 2.15 (s, 9H), 6.45 (d, 6H, J_(PH) =13.5 Hz); ³¹ P NMR(CDCI₃, 121.5 MHz) δ-2.25; MS m/z 241 (M-CH₂ OAc)⁻.

Synthesis of Phenylphosphonate Bis(acetoxy-methyl)ester(3)--Phenylphosphonic acid (31.6 mg, 0.2 mmol) and diisopropylethylamine(DIEA, 130 mg, 1.0 mmol) were dissolved in 1 mL dry CH₃ CN. AM-Br (77mg, 0.5 mmol) was added and the solution was stirred at room temperatureover night. After evaporation of the solvent the solid residue wasextracted with dry toluene. Purification of the crude product 3 wasperformed on a Si60 column (10×40 mm) with 75% toluenel 25% ethylacetate to yield 52 mg 3 (86%) as a clear oil. ¹ H NMR (CDCI₃, 200 MHz)δ 1.95 (s, 6H), 5.66 (dAB, 4H, J_(AB) =5.3 Hz, J_(PH) =13.8 Hz, --CH₂--), 7.30-7.55 (m, 3H), 7.70 (m, 2H). ³¹ P NMR (toluene-d₈, 121.5 MHz) δ18.70.

Synthesis of N⁶,O^(2') -Dibutyryl Adenosine 3',5'-cyclic MonophosphateAcetoxymethyl Ester --bt₂ cAMP/AM (4a/4b)--Two different methods wereused to synthesize bt₂ cAMP/AM. Method A: The sodium salt of N⁶,O^(2')-dibutyryl cAMP (12.5 mg, 25 μmol) was dissolved in 1 mL MeOH-H₂ O (1:1)and passed through a Dowex 50W-X8 column (10×40 mm, H⁺ -form). The freeacid was eluted with 15 mL 50% MeOH. After evaporating to dryness, DIEA(6 mg, 50 μmol) and 1 mL dry CH₃ CN were added. The reaction was startedby the addition of AM-Br (16 mg, 94 μmol). After stirring the solutionat room temperature for 4 days, the reaction mixture was chromatographeddirectly on a Si60 column (10×40 mm, 230-400 mesh) with 95% CH₃ CN/5%hexane as the eluent under slight pressure. The eluant was collected in5 mL fractions. Fractions 5-7 contained 5.3 mg (38% yield) of the fastereluating diastereomer of dibutyryl cAMP acetoxymethyl ester (4a) in highpurity. ¹ H NMR (CDCI₃, 300 MHz) δ 1.05 (t, 3H, J=7.0 Hz), 1.12 (t, 3H,J=7.0 Hz), 1.74 (m, 2H), 1.84 (m, 2H), 2.20 (s, 3H), 2.51 (m, 2H), 2.95(t, 2H, J=7.0 Hz), 4.36 (ddd, 1H, J=2.7, 10.1, 10.1 Hz, H4'), 4.49 (dd,1H, J=10.0, 10.0 Hz, H5'_(ax)), 4.66 (dddd, 1H, J=2.7, 10.0, 10.0, 22.1Hz, H5'_(eq)), 5.67-5.95 (m, 4H, --CH₂ --, H2', H3'), 6.04 (s, 1H, H1'),8.01 (s, 1H, H2), 8.49 (broad s, 1H, N⁶ H), 8.78 (s, 1H, H8); ³¹ P NMR(CDCI₃, 121.5 MHz) δ-5.0 ppm.

Fractions 8+9 yielded 8.7 mg of a clear oil which containeddiisopropyl-ethylammonium bromide and the slower eluting diastereomer of4b (2:1 w/w as determined by NMR, yield 2.9 mg 4b, 21% fromdibutyryl-cAMP). ¹ H NMR (CDCI₃, 200 MHz), δ 0.99 (t, 3H, J=7.0 Hz),1.05 (t, 3H, J=7.5 Hz), 1.70 (m, 4H), 2.18 (s, 3H), 2.45 (t, 2H, J=7.0Hz), 2.89 (t, 2H, J=7.5 Hz), 4.40-4.70 (m, 3H, H4', H5'_(eq), H5'_(ax)),5.62-5.78 (AB-part of ABX, 2H, J_(AB) =5.1 Hz, --CH₂ --), 5.83 (m, 2H,H2', H3'), 6.01 (s, 1H), 8.02 (broad s, 1H, H2), 8.51 (broad s, 1H, N⁶H), 8.69 (s, 1H, H8); ³¹ P NMR (CDCI₃, 121.5 MHz) δ-8.0 ppm; MS (4a/4b1:1 mixture) m/z (M+H)⁺ calcd 542.1652, obsd 542.1681.

Method B: 58 mg (0.12 mmol) of the sodium salt of Bt₂ cAMP was dissolvedin 0.5 mL H₂ O and 300 μL 1.8M AgNO₃ solution was added. The resultingwhite precipitate was filtered off washed with H₂ O, and dried to yield30.5 mg (45%, 54 μmol) of the silver salt of Bt₂ cAMP. The white powderwas suspended in 1 mL of dry CH₃ CN and 51 mg (330μmol) AM-Br wereadded. The suspension was frequently sonicated for 4h at roomtemperature. The two resulting diastereomeric acetoxymethyl esters 4aand 4b were purified as described under method A to yield 2.8 mg of thefast eluting isomer 4a (10% yield) and 9.6 mg (35%) of the slow elutingdiastereomer 4b. NMR and MS analysis of the products of both methodswere identical.

Synthesis of N²,O^(2') -Dibutyryl Guanosine 3',5'-cyclic MonophosphateAcetoxymethyl Ester bt₂ GMP/AM (5at5b)--The sodium salt of Bt₂ cGMP (24mg, 47 μmol) was passed through Dowex 50W-X8 (H⁺ form) and the free acidwas eluted with 15 mL 50% MeOH. After evaporating to dryness, 1 mL dryacetonitrile, 13 mg (100 μmol) DIEA and 21 mg (135 μmol) AM-Br wereadded. The solution was stirred overnight, evaporated to dryness,dissolved in CH₃ CN/hexane (95:5, v/v) and eluted over a Si60 column(10×40 mm) to yield 11 mg (40%) of the two diastereomers of dibutyrylcGMP-AM (5a/5b) as a mixture. ¹ H-NMR (5a only, CDCI₃, 200 MHz) δ 1.00(m, 6H), 1.74 (m, 4H), 2.38 (s, 3H), 2.42 (m, 2H), 2.48 (m, 2H), 4.18(ddd,1H, J=4.0, 10.0, 10.0 Hz, H4'), 4.30-4.54 (m, 2H, H5'_(ax),H5'_(eq)), 5.13 (ddd, 1H, J=1.8, 4.1, 10.0 Hz, H3'), 5.56 (dd, 1H,J=4.1, 4.1 Hz, H2'), 5.71 (dAB, 2H, J=12.5, 9.1 Hz, --CH₂ --), 6.04 (d,1H, J=4.0 Hz, H1'), 7.65 (broad s, 1H, N² H), 10.14 (s, 1H, H8), 12.30(broad s, 1H, N¹ H); ³¹ P NMR (CDCI₃, 121.5 MHz) δ-5.5 and -8.5 ppm; MSm/z (M+H)⁺ calcd 558.1601, obsd 558.1611.

The most general and economical synthetic route to acetoxymethylphosphate esters is believed to be alkylation of the parent phosphateanions by acetoxymethyl halides. The instability of acetoxymethanolprecludes its phosphorylation. Preliminary synthetic attempts, similarto the experiments of Srivasta and Farquhar (15), were performed on4-methylumbelliferyl phosphate and phenyl phosphonate as readilyavailable model compounds detectable by UV absorption and bearing nocompeting nucleophilic centers. 4-Methylumbelliferyl phosphatebis(acetoxymethyl)ester (Table I-1) was successfully prepared in 73%yield by suspending the disilver salt of 4-methylumbelliferyl phosphatein dry acetonitrile, adding acetoxymethyl bromide (AM-Br) (38), andsonicating the heterogeneous mixture at frequent intervals for 24 hours.The ¹ H NMR of the supernatant showed an AB doublet at 5.7 ppm for themethylene group of the acetoxymethyl ester, a typical pattern for allphosphate acetoxymethyl esters reported here. The synthesis of phosphatetris(acetoxymethyl)ester (Table I-2) offered a possibility to directlymonitor the progress of the reaction. Yellow Ag₃ PO₄ was reacted withAM-Br as described above. Disappearance of the color after 36 hoursindicated completion of the reaction. The product was the only compoundin the organic phase (98% yield).

An alternative to silver salts is desirable for polyphosphates ormolecules bearing oxidizable functionalities. Direct treatment ofphenylphosphonic acid with an excess of the hindered basediisopropylethylamine (DIEA) and AM-Br eventually gave an 86% yield ofthe phenylphosphonate bis(acetoxy-methyl)ester (Table I-3).

Analogous reactions worked, albeit in lower yield, for N⁶,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate acetoxymethyl ester (Bt₂cAMP/AM,4a/4b) and N²,2'-O-dibutyryl guanosine 3',5'-cyclicmonophosphate acetoxymethyl ester (Bt₂ cGMP/AM, 5a/5b). The commerciallyavailable sodium salts of Bt₂ cAMP and Bt₂ cGMP were converted into thefree acids on Dowex 50W-X8 and then into DIEA salts. Reaction took placein dry CH₃ CN with an excess of DIEA and AM-Br for 5 days at roomtemperature. Both nucleotide AM-esters were purified on silica gel 60(CH₃ CN/hexane 19:1 v/v) after evaporation of the solvent. The twodiastereomers of Bt₂ cAMP/AM (4a/4b) were isolated in yields of 37% and21% for the fast and slow-eluting isomers, the latter co-eluting withresidual DIEA. ³¹ P-NMR resonances were -5.0 ppm and -8.0 ppm,respectively, but absolute configurations were not determined. Theanalogous two diastereomers of Bt₂ cGMP/AM (5a/5b) could not beseparated under the described conditions, but were free of DIEA. Bt₂cAMP/AM was also prepared by alkylating the silver salt of Bt₂ cAMP withAM-Br in CH₃ CN with frequent sonication for 24 h. These heterogeneousconditions reversed the enantiomeric preference, giving the fast andslow-migrating isomers in 10% and 35% yields.

The synthesis of cAMP and cGMP described above utilizes butyryl groupsto mask or protect the hydroxyl groups. This is because the directconversion of a cAMP or cGMP into their acetoxy esters yields onlyminute amounts of product. Trimethylsilyl (TMS) groups were also foundto work well as a protective group. The synthesis schemes for direct andTMS protected synthesis is shown in FIG. 8.

Synthesis of the acetoxy ester of cAMP using TMS is set forth below:

General Methods--Proton and ³¹ P NMR spectra were obtained in CDCI₃ withresidual CHCI₃ (δ=7.26), being used as the internal standard for ¹ Hspectra. 85% phosphoric acid was used as an external standard for ³¹ Pspectra. All NMR spectra were recorded on either a Varian Gemini-200(200 MHz) or a General Electric QE-300 (300 MHz) spectrometer and arereported with the same abbreviations as in the preceding examples.

Acetonitrile were stored over activated molecular sieve (3 Å) for atleast 3d. All other solvents were purchased in highest purity availableand were used as received. N,N-Diisopropylethylamine (DIEA) wasdistilled from CaH₂. Acetoxymethyl bromide (AM-Br) was preparedaccording to known procedures. cGMP was from ICN or Calbiochem. Allother nucleotides were from Sigma. All other reagents were from Aldrich.

The following three step synthesis is outlined in FIG. 4.

Synthesis of 2'-O-Trimethylsilyl Adenosine 3',5'-cyclic Phosohate (FIG.4A)--The free acid of cAMP (50 mg, 0.15 mmol) was suspended in 3 mL dryDIEA. One (1) mL Hexamethyidisilazane (HMDS) and 0.5 mL Trimethylsilylchloride (TMS-CI) were added under Ar and the mixture was heated to 100°C. for 3 hours. After cooling to r.t. all volatile components wereevaporated off in high vacuum. The residual oil was extracted with 2×2mL dry toluene. A sample of the extract, exclusively containingN⁶,2'-O-di(trimethylsilyl) adenosine 3',5'-cyclic monophosphatetrimethylsilyl ester, was evaporated to dryness and analyzed by NMR: ¹ HNMR (toluene-dg, 200 MHz) diastereomer 1 (80%) δ 0.32 (s, 9H), 0.45 (s,9H), 0.47 (s, 9H), 4.15-4.61 (m, 3H, H4', H5'_(eq), H5'_(ax)), 4,98 (d,1H, J=4.7 Hz, H2'), 5.60 (s, 1H, N⁶ H), 5.74 (ddd, 1H, J=1.6, 4.7, 9.5Hz, H3'), 5,83 s, 1H, H1', 7.64 (s, 1H, H2), 8.6 (s, 1H, H8).Diastereomer 2 (20%) δ 4.65 (d, 1H, J=4.2 Hz, H2'), 5.20 (m, 1H, H3'),5.66 (s, 1H, N⁶ H); 6.05 (s, 1H, H1'), 7.77 (s, 1H, H2), 8.58 (s, 1H,H8). The toluene extract was treated with 12 μL MeOH (0.3 mmol) for 3min. followed by rapid evaporation of the solvents. The remaining whitesolid fairly pure 2'-O- TMS-cAMP (1). ¹ H NMR (CD, OD, 200 MHz) δ 0.80(s, 9H, TMS), 4.32 (m, 3H, H4', H5'), 4.65 (d, 1H, J=8.2 Hz, H2'), 4.95(m, 1H H3'), 6.12 (s, 1H, H1'), 7.16 (broad s, 2H, NH₂), 8.40, 8.45 (2s,1H each, H2, H8). ³¹ H-NMR O.

Synthesis of 2'-O-Trimethylsilyl Adenosine 3',5'-cyclic MonophosphateAcetoxymethyl Ester (FIG. 4-B)--25 mg (0.062 mmol) 2'-TMS-cAMP (1) wasdissolved in 0.5 mL dry CH₃ CN containing 0.05 mL DIEA (0.28 mmol) underAr and 0.028 mL (0.28 mmol) AMBr were added. The mixture was stirred for3 days at r.t. then evaporated to dryness. The crude produce waspurified on a Si60 column (4×1.5 cm) with dry CH₃ CN saturated withhexane as the eluent. Prior to the separation the column was washed withthe same eluent containing 0.1% acetic acid followed by the eluentalone. Most of the 2'-O-TMS-cAMP/AM (2) eluated just before HDIEA⁺ Br toyield 20.5 mg (0.042 mmol, 68%). The product consisted of 90% of one ofthe R_(P) /S_(P) -diastereomers, as determined by ³¹ P NMR. ¹ H NMR(CDCI₃, 300 MHz) δ 0.22 (s, 9H, TMS), 2.16 (s, 3H, --OCCH₃), 4.43 (m,2H, H4', H5'_(ax)), 4.64 (m, 1H, H5'_(eq)), 4.85 (d, 1H, J=5.2 Hz, H2'),5.34 (m, 1H, H3'), 5.74 (d, 2H, J=13.1 Hz, --CH₂ --OAc), 4.86 (broad s,2H, NH₂), 5.91 (s, 1H, H1'), 7.87 and 8.41 (2s, 1H each, H2 and H8). ³¹P NMR (CDCI₃, 121.5 Hz) δ-7.58 (90%), -4.62 (10%).

Synthesis of Adenosine 3',5'-cyclic Monophosphate Acetoxymethyl Ester(FIG. 4-C)--14 mg (0.029 mmol) of 2 were dissolved in 1 mL of a 1:1(v/vO mixture of CHCI3/CH₃ CN and 2 μL HF (49%) was added. The mixturewas gently swirled for 2 min. before the solvents were evaporated off toquantitatively yield cAMP/AM (3) as a white solid. ¹ H NMR (CD₃ OD, 300MHz) δ 2.17 (s, 3H, --OAc), 4.48 (ddd, 1H, J=3.6, 9.5, 9.5 Hz, H4'),4.50 (m, 1H, H5'_(ax)), 4.74 (m, 1H, H5'_(eq)), 5.33 (ddd, 1H, J=1.0,4.8, 12.3 Hz, H3'), 5.75 (m, 2H, --CH₂ --OAc), 6.15 (s, 1H, H1'), 8.41and 8.42 (2s, 1H each, H2, H8). ³¹ P NMR (CD₃ OD, 121.5 MHz) δ-6.85.

The following biological tests were conducted to demonstrate thebiological activity of the cAMP derivative after it passes into thecell.

Activation of intracellular protein kinase A. The premier target of cAMPin most cells is the cAMP-dependent protein kinase (PKA) (45). To showthat this enzyme can be activated by extracellular application of Bt₂cAMP/AM, we used a recently-developed assay for PKA activation in singlecells (39). When PKA is doubly labeled with fluorescein on its catalyticsubunits and rhodamine on its regulatory subunits to produce FICRhR,fluorescence energy transfer from the fluorescein to rhodamine occurs inthe holoenzyme complex but is disrupted upon activation and dissociationof the subunits. The change in the ratio of fluorescein to rhodamineemissions parallels the increase in kinase activity and can benondestructively imaged in single cells. REF-52 fibroblasts weremicroinjected with FICRhR and emission ratio images recorded at roomtemperature as previously described (27). 30 min after injection, 0.1,1, or 10 μM Bt₂ cAMP/AM were added extracellularly (FIG. 1-Graph A). Thehighest dose yielded a maximal change in fluorescence ratio within 15min. The intermediate dose gave a shallower slope and a lower plateau toslightly over 50% of the maximal change. The onset of the separation ofregulatory and catalytic subunit of FICRhR occurred roughly 2 min afterthe addition of the cAMP derivative. Much the same delay and overalltime course occurred with nonesterified Bt₂ cAMP, though much higherconcentrations, 1 mM, were required (FIG. 1-Graph B). Other widely used,supposedly lipophilic CAMP derivatives showed no delay in beginning toactivate PKA, but millimolar concentrations were still required (FIG.1-Graphs C & D).

To show that intracellular enzymatic hydrolysis of the ester groups isrequired we examined the binding properties of Bt₂ cAMP/AM and Bt₂ cAMPto FICRhR in vitro. The highest concentration of Bt₂ cAMP/AM used in theother assays (10 μM) gave no separation of the subunits, while Bt₂ cAMPwas roughly 1/100 as potent as cAMP probably due to contamination by 1%monobutyryl-cAMP as specified by the supplier (Sigma) (See Table II).

                  TABLE II    ______________________________________    In-vitro cAMP-dependent kinase activation assay.              cAMP      Bt.sub.2 cAMP.sup.a                                 Bt.sub.2 cAMP/AM    ______________________________________    concentration  μM!                1     10     200.sup.b                                  10   10  10    % kinase.sup.c activation                67    91     100  16   61  0    ______________________________________     .sup.a The slight residual activity of Bt.sub.2 cAMP is probably due to a     impurity of N.sup.6 -monobutyryl cAMP (1%) as specified by the supplier.     .sup.b 200 μM cAMP was considered the maximal dose necessary to fully     dissociate FICRhR.     .sup.c Labelled cAMPdependent kinase type I (FICRhR). This labelled     isoform is more stable against subunit dissociation in the absence of cAM     at the low enzyme concentrations used in this assay than labelled type II

One of the many well-known cell functions controlled by cAMP isintestinal transepithelial CI⁻ -secretion (46). A convenient test systemis the intestinal cell line T₈₄, in which chloride secretion can becontinuously monitored by mounting confluent monolayers of cells inUssing chambers (42). FIG. 2A shows the CI⁻ -secretion measured as shortcircuit current (I_(SC)) across the cells. The addition of Bt₂ cAMP/AMat a concentration of 3 μM to the serosal bathing solution caused anincrease in I_(SC) with a maximum after 20 min. Higher concentrations ofthe derivative caused faster but not significantly greater responses,whereas lower concentrations reached lower maximum I_(SC) values. TheI_(SC) values obtained at an arbitrary intermediate time, 12 min afteraddition of various cAMP-derivatives, were used to determine the dosedependency (FIG. 2B). The dose response curves were parallel, with EC₅₀values of 2 μM and 400 μM for Bt₂ cAMP/AM and Bt₂ cAMP respectively.Therefore the introduction of the acetoxymethyl group on the phosphateincreased the potency by 200 fold in this assay by circumventing thepermeability barrier. Furthermore, the acetoxymethyl ester seems to becleaved inside T₈₄ cells without significant delay, since the two agentsgave essentially indistinguishable kinetics of activation. Tests withthe cAMP-derivatives 8-Br-cAMP and 8-pCPT-cAMP showed activation of CI⁻-secretion with EC₅₀ values of 1.5 mM and 100 μM, respectively.

Fish dermal chromatophores exhibit a tightly regulated movement ofpigment granules either inward into a highly aggregated central mass, oroutward, dispersing the pigment throughout the cell. In angelfish(Pterophyllum scalare) melanophores this movement is microtubule basedand cAMP regulated (44,47) but relatively refractory to external cAMPanalogs. Melanophores permit a visual single-cell assay for the abilityof cAMP analogs to enter cells and mimic cAMP actions. The melanophoreswere isolated on angelfish scales and the epidermis was stripped off.The 60-100 melanophores per scale were pretreated with an α₂ -adrenergicagonist to reduce endogenous cAMP and start with full aggregation.Extracellular Bt₂ cAMP/AM then caused dispersion of the pigment in adose-dependent manner (FIG. 3A). A concentration of 100 μM Bt₂ cAMP/AMwas enough to cause essentially complete dispersal; however, 1 mM gave aslightly faster onset of action and could not be readily reversed byremoval of the extracellular Bt₂ cAMP/AM and administration ofepinephrine, whereas the effect of 100 μM was easily reversed (FIG. 3B).Dispersion was just detectable at 1 μM and half-maximal near 10 μM (FIG.3A). By comparison, 1 mM Bt₂ cAMP was unable to cause any detectabledispersion. Hence the AM ester group increased the potency by more than1000 in this assay. The effectiveness of Bt₂ cAMP/AM shows that theinertness of Bt₂ cAMP in melanophores is due to poor permeability ratherthan susceptibility of Bt-cAMP to phosphodiesterases or selectivity of akinase binding sites for cAMP substitution (48). The preceding examplesare summarized in (64).

The preceding strategy for increasing permeability of cAMP and cGMP,i.e. acetoxymethyl esterification in combination with butyryl groupmasking of the hydroxyl groups, was found not to be effective forinositol phosphates. Acetoxymethyl esters of inositol triphosphateswhich had their hydroxy groups blocked with butyryl groups were found,to have relatively low potency, perhaps because butyryl esters locatedbetween bulky phosphate groups cannot be cleaved by intracellularenzymes. However, if the hydroxyl groups were left unesterified and thephosphates were esterified with acetoxymethyl groups, the resultingcompounds had insufficient membrane permeability to enter cells. Wefound that, in addition to leaving the hydroxyl groups unesterified, itwas also necessary to replace the acetoxymethyl groups with morehydrophobic groups in order to provide acceptable cell permeability andsubsequent ester cleavage. We discovered that more hydrophobic esterssuch as, propionyloxymethyl(PM) esters (R=ethyl, R'=H) orbutyryloxymethyl(BM) esters (R=propyl, R'=H), permeate the cell membraneat higher rates while still being amenable to cleavage upon entry intothe cell.

Inositol phosphate esters in accordance with the present invention havethe formula ##STR10## wherein A₁ to A₆ is H, OH, F or ##STR11## whereinR is an alkyl group having from 2 to 6 carbon atoms and R' is H or CH₃or R is CH₃ and R' is CH₃ and wherein at least one of A₁ to A₆ is aphosphoester having the formula set forth above.

Preferred esters are the propionyloxymethyl and butyryloxymethyl estersof: inositol-1,4,5-triphosphate; 3-deoxy-inositol-1,4,5-triphosphate;3-fluoro-inositol-1,4,5-triphosphate; inositol-1,3,4-triphosphate;inositol-1,3,4,5-tetraphosphate; and inositol-3,4,5,6-tetraphosphate.

Examples of synthesis and use of preferred exemplary inositolpolyphosphate esters in accordance with the present invention is asfollows:

The general synthesis scheme which may be used to make a number ofdifferent esters in accordance with the present invention is set forthin FIG. 5.

The synthesis of phosphotriesters with neighboring unprotected hydroxylsis difficult because the phosphates very easily migrate or cyclize intothe free hydroxyls. The key is to use benzyl ethers to protect thehydroxyls for as many steps as possible and to find conditions to removethe benzyls at the last stage without affecting the phosphateacyloxyalkyl esters (FIG. 5). Thus in the synthesis of, for example,inositol triphosphate propionyloxymethyl ester (IP₃ /PM), the hydroxylson the 2, 3, and 6-positions are initially protected as benzyl groups.Phosphorylation of 1 withbis(β-cyanoethyl)N,N-diisopropylphosphoramidite, oxidation of thephosphites, and ammonolysis of the cyanoethyl protecting groups affords2,3,6-tri-O-benzyl-IP₃ (2a). Esterification with bromomethyl propionate(3, R=Et, R'=H) gives fully protected inositol trisphosphate (4a).Finally, catalytic hydrogenolysis over palladium acetate in acetic acidprovides IP₃ /PM (5a, R=Et, R'=H) without phosphate migration orcyclization. This general procedure may be used to synthesize othermembrane-permeant, intracellularly hydrolyzable esters of IP₃ and otherimportant inositol polyphosphates such as 3-deoxy-1,4,5-IP₃,3-fluoro-1,4,5-IP₃, 1,3,4-IP₃, 1,3,4,5-IP₄, and 3,4,5,6-IP₄.

A detailed description of the synthesis is as follows:

D-2,3,6-tri-O-benzyl-myo-inositol 1a and D-2,6-di-O-benzyl-myo-inositol1d, which are known compounds (65,66), are synthesized from myo-inositolby a modified procedure (for 1a) or as published (for 1d).D-2,3-di-O-benzyl-3-deoxy-myo-inositol 1b andD-2,3-di-O-benzyl-3-deoxy-3-fluoro-myo-inositol 1c, may be prepared intwo steps from known precursors (67,68),D-1,4,5-tri-O-benzoyl-6-O-benzyl-3-deoxy-myo-inositol (compound 10 inref. 67) orD-1,4,5-tri-O-benzoyl-6-O-benzyl-3-deoxy-3-fluoro-myo-inositol (compound5b in ref. 68) respectively. These starting materials may be benzylatedwith benzyl trichloroacetimidate in the presence of a catalytic amountof trifluoromethanesulfonic acid. Removal of the benzoyl protectinggroups with potassium carbonate in methanol overnight affords 1b and 1c,respectively. The experimental procedure outlined in FIG. 5 is similarfor 5a, 5b, 5c and 5d. Here we use 5a as an example. All reactions aredone under an atmosphere of argon unless otherwise specified.

D-2,3,6-tri-O-benzyl-myo-inositol-1,4,5-triphosphate (2a), ammoniumsalt: 65 mg 1a (0.144 mmol) was dissolved in 1 ml dichloromethane and 1ml acetonitrile. Bis(β-cyanoethyl)-N,N-diisopropylphosphoramidite(69)(180 μl, 0.65mmol) was added, followed by 48 mg tetrazole (0.69mmol) dissolved in 1.2 ml acetonitrile. After stirring at roomtemperature for 2 hours, the reaction flask was cooled in an ice bath.An excess of t-butyl hydroperoxide (300 μl of a 5M solution indichloromethane) was added in one portion. Thirty minutes later, the icebath was removed and stirring was continued at room temperature foranother hour. The solvent was removed under reduced pressure. Theremaining syrup was resuspended in the minimum amount ofdichloromethane/methanol (12:1). 130 mg of a clear glass was obtained.This material was dissolved in 1.5 ml methanol and mixed with 6 mlconcentrated ammonium hydroxide. It was refluxed at 60° C. for 3 hoursand the solvent was removed under reduced pressure. The remainingmaterial was used directly for the next step without furtherpurification.

D-2,3,6-tri-O-benzyl-myo-inositol-1,4,5-triphosphate propionyloxymethylester (4a). The above material (2a) was suspended in 1 ml acetonitrileand 0.2 ml diisopropylethylamine (DIEA). It was then sonicated for ashort interval and the solvent was removed under reduced pressure. Thisprocess was repeated a few more times until a homogeneous solution wasobtained after adding acetonitrile and DIEA, indicating that thecounterions for the phosphate groups had been exchanged from ammonium todiisopropylethylammonium. An excess (˜150 μl) of bromomethyl propionate(3, R=Et, R'=H, prepared as in ref. 70) was then added and left to reactfor 48 hours. The solvent was removed under vacuum and the remainingsyrup was first purified by silica gel flash column chromatography,eluting with ethyl acetate. The pale yellow glass obtained uponevaporation was further purified by HPLC on a C18 reverse-phase column,eluting with 78% methanol in water. 15 mg clear glass was obtained, 10%overall yield from 1a. ¹ H-NMR (CDCI₃): δ=1.1 (m, 18H), 2.2-2.45 (m,12H), 3.5 (dd, 1H, J=12 Hz, 2 Hz), 4.05 (t, 1H, J=11.2 Hz), 4.3-4.5 (m,3H), 4.7-4.9 (m, 7H), 5.3-5.7 (m, 12H), 7.2-7.5 (m, 15H), FAB MS m/z(M+Cs)⁺. Calculated: 1339.2293. Observed: 1339.2261.

D-myo-inositol-1,4,5-triphosphate propionyloxymethyl ester (5a, R=Et,R'=H). 15 mg 4a was dissolved in 2 ml glacial acetic acid. 20 mgpalladium acetate and a catalytic amount of trifluoroacetic acid wasadded. The mixture was stirred under 1 atm hydrogen gas at a temperaturebelow 20° C. to minimize phosphate triester migration. After 4 hours ofhydrogenation, the catalyst was filtered off and the acetic acid waslyophilized. 10 mg clear glass was obtained. ¹ H-NMR (CD3OD): δ=1.2 (m,18H), 2.4 (m, 12H), 3.7 (dd, 1H, J=9.9 Hz), 2.4 Hz), 4.0 (t, 1H, J=9.3Hz), 4.2-4.4 (m, 3H), 4.65 (q, 1H, J=8.4Hz), 5.7 (m, 12H).

D-myo-inositol-1,4,5-triphosphate butyryloxymethyl ester (5a, R=Pr,R'=H) was prepared identically but with bromomethyl butyrate (3, R=Pr,R'=H) in place of bromomethyl propionate. ¹ H-NMR (CD3OD): δ=0.95 (m,18H), 1.7 (m, 12H), 2.4 (m, 12H), 3.7 (dd, 1H, J=9.9 Hz, 2.4 Hz), 4.0(t, 1H, J=9.3 Hz), 4.2-4.4 (m, 3H), 4.65 (q, 1H, J=8.4Hz), 5.7 (m, 12H).FAB MS m/z (M+Cs)⁺. Calculated: 1153.1824. Observed: 1153.1850.

Biolocical Applications of IP₃ /PM and IP₃ /BM

The best-established biological effect of IP₃ is to release Ca²⁺ frominternal stores (26), so IP₃ /PM (5a, R=Et, R'=H) was tested by imagingcytosolic free Ca²⁺ levels in single REF-52 fibroblasts using standardmethodology (58). Cells were loaded with the Ca²⁺ -indicator fura-2 andviewed by fluorescence excitation ratioing. IP₃ /PM (40 μM) was appliedextracellularly while monitoring cytosolic Ca²⁺ concentrations. As shownin FIG. 6, the Ca²⁺ concentration increased rapidly after addition ofIP₃ /PM (vertical dotted line labeled 1). Subsequent addition ofvasopressin (vertical dotted line labeled 2), a well-known Ca²⁺-releasing hormone (59) had much less effect than normal. This occlusiondemonstrates that IP₃ /PM had already partially exhausted the samepathways to elevate Ca²⁺ as utilized by a physiological agonist,vasopressin.

Biological tests on 1321N1 astrocytoma cells were performed similarly tothe tests on REF-52 cells (71). The results of the tests are shown inFIG. 7. The concentration of Cytosolic free Ca²⁺ (ordinate, in units ofμM) in fura-2-loaded 1321N1 astrocytoma cells is shown as a function oftime (abscissa, in units of seconds). At the dotted vertical linelabeled 1, 20 μM inositol-1,4,5-triphosphate propionyloxymethyl ester(5a, R=Et, R'=H) was added. A sizable elevation of cytosolic Ca²⁺ wasobserved after about 100 second delay. Subsequently the cells couldrespond to carbachol (200 μM, delivered at the dotted vertical linelabeled 2), a drug that stimulates the endogenous generation ofinositol-1,4,5-triphosphate. However, the response to carbachol isdepressed by the prior treatment with the inositol-1,4,5-triphosphatepropionyloxymethyl ester. Likewise the Ca²⁺ response to thapsigargin(100 nM, delivered at the dotted vertical line labeled 3), another drugthat releases stored Ca²⁺, is depressed.

A test similar to the one shown in FIG. 7 was conducted except that themedium lacked extracellular Ca²⁺ and a dose of 60 μMinositol-1,4,5-triphosphate propionyloxymethyl ester was added at thedotted vertical line labeled 1. The results of the test are shown inFIG. 8. A sizable elevation of cytosolic Ca²⁺ was observed after about a50 second delay. Because no extracellular Ca²⁺ was present, thisresponse must have represented release from intracellular Ca²⁺ stores.Subsequently the cells' response to carbachol (200 μM, delivered at thedotted vertical labeled 2) was greatly depressed by the prior treatmentwith the inositol-1,4,5-triphosphate propionyloxymethyl ester. The muchgreater degree of inhibition of the carbachol response compared to theprevious example (FIG. 7) is due to the higher dose of theinositol-1,4,5-triphosphate propionyloxymethyl ester and the absence ofextracellular Ca²⁺, which reduces refilling of Ca²⁺ stores.

In a further example, the same test as set forth above was conductedwith astrocytoma cells, except that 2 μM inositol-1,4,5-triphosphatebutyryloxymethyl ester (5a, R=Pr, R'=H) was used instead of thepropionyloxymethyl ester. The test results are shown in FIG. 9. Thebutyryloxymethyl ester is somewhat more potent but its effects areslower to appear and decay.

The above biological results show that neutral, hydrophobic,membrane-permeant derivatives of inositol phosphates can be synthesizedand show the expected biological activity when applied extracellularlyto intact REF-52 fibroblasts and astrocytoma cells. IP₃ /PM releasedCa²⁺ from internal stores, as would be expected for an agent thatmimicked IP₃.

The above membrane-permeant derivatives of inositol polyphosphatesutilize acyloxy groups esterifying all the phosphates to mask theirnegative charges. After crossing the plasma membrane, the compoundsgradually hydrolyze inside the cell. As a result, both inositolphosphate release and the subsequent internal calcium release developswith a sigmoidal time course. The accumulation of active species can belimited because inositol phosphate may be destroyed by metabolic enzymesat rates comparable to its release from the protected form. Furthermore,if the hydroxyl groups are left free and unprotected, migration ofphosphate esters to vicinal hydroxyls, reduction of the efficiency ofdelivery, and loss of pharmacological specificity may occur. Caging ofthe membrane-permeant inositol phosphate esters provides at least twoimprovements. First, all the hydroxyl groups are blocked by protectinggroups, eliminating phosphate migration and increasing the efficiencyand isomeric specificity of inositol phosphate delivery. Second, afterentering the cell and undergoing hydrolysis, the caged inositolphosphate remains inactive because a key hydroxyl group, the 6-hydroxyl,is caged by a photolabile protecting group. This group should also blockmetabolic degradation, thereby helping caged IP₃ (structure 5 in FIG.10) accumulate inside cells. Upon uncaging with UV light, the cagedcompound releases active species that cause a sudden Ca²⁺ !_(i) increasemimicking the one generated by physiological agonist stimulation.

The above-described caged inositol polyphosphate esters in accordancewith the present invention have the following formulas: ##STR12##Wherein R₁ is H, --CHO, COOCH₃ or --COCH₃ ; R₂ is H or --P(O)(OR₄)(XR₄);or R₁ and R₂ together are --CH₂ --, --CHMe--, --CMe₂ --, --CH(OMe)--,--CMe(OMe)-- or --C(OMe)₂ --; R₃ is a photolabile protecting group;

R₄ ##STR13## ; and

X is O or S.

When R₁ and R₂ taken together are --CH₂ --, the compound has the formula##STR14## Likewise, when R₁ and R₂ taken together are --CH(OMe)--, thecompound has the formula: ##STR15## Preferred compounds are those whereX is O.

The photolabile protecting group (R₃) may be any of the known compoundswhich are used to provide photolabile protection of reactive sites (seeReference 73). Exemplary photolabile protecting groups (R₃) include R₅,--CH₂ OR₅ or --COOR₅ where R5 is ##STR16## R₆ =H, Me, CH₃ CO and R₄ isas defined previously.

Examples of synthesis and use of the above identified caged compounds isas follows:

The general synthetic route to a preferred exemplary caged compound(D-IP₃ /PM) is outlined in FIG. 10. A key step is to protect the6-hydroxyl of inositol, which plays a critical role in binding to theIP₃ receptor and releasing calcium, with the photoliable group4,5-dimethoxy-2-nitrobenzyl (DMNB). The 2- and 3-positions are coveredby an orthoformate. Phosphorylation of 4 and esterification of theresulting trisphosphate 5 (caged IP₃) with bromomethyl propionateafforded IP₃ /PM. The strategy is general enough to make other cagedmembrane-permeant IP₃ derivatives with different substituents on 2- and3-position. In addition, this general procedure allows replacement ofthe phosphate by phosphothioate, which is metabolically more stable andhas potential in drug development.

The 2-nitrobenzyl group and its derivatives 1-(2-nitrophenyl)ethyl,4,5-dimethoxy-2-nitrobenzyl, and α-carboxy-4,5-dimethoxy-2-nitrobenzyl,have been widely used in caging biomolecules. These groups mask thecharged (i.e. carboxylate or phosphate) or polar (i.e. amine orhydroxyl) functionalities of the molecules and increase theirhydrophobicity, often increasing their membrane permeability. Beforephotolysis, these caged compounds are required to be biologicallyinactive because at least one of the key functionalities is blocked.However, the activity of the molecule can be triggered by a pulse of UVlight (360 nm) because the nitrobenzyl group and its derivatives arephotolabile. This strategy is very valuable for in vivo biologicalapplication. It allows control of the onset of bioactivity in livingcells with millisecond temporal precision. The mechanism of thisphoto-transformation and the applications of caging compounds have beenreviewed (72,73). A few examples of caged molecules which have hadsuccessful applications in biology include caged cAMP (74,75), cagednitric oxide (76,77), caged fluorescein (78), caged calcium (79,80),caged glutamate (81-83), and caged IP₃ (84).

Commercially available caged inositol triphosphate (IP₃) has one of itsvicinal phosphates (either the 4- or 5-phosphate, both of which playcritical roles in binding to the IP₃ R) esterified by a nitrophenylethylgroup (85). The molecule still has over 3 negative charges atphysiological pH and is membrane-impermeant, so that it can only beintroduced into cells by techniques that disrupt their plasma membranes.The new caged and membrane-permeant IP₃ derivatives of the presentinvention have all phosphates covered by acyloxy asters and all thehydroxyls concealed by suitable protecting groups.

Among the commonly-used caging groups, 4,6-dimethoxy-2-nitrobenzyl(DMNB) group is preferred as the protecting group for the 6-hydroxyl.Compared to other photolabilo groups such as nitrobenzyl (NS) ornitrophenylethyl (NPE), the absorbance maximum of DMNB is at longerwavelengths (360 nm). Even though the quantum yield of DMNB for thephotorelease was relatively low, its relatively high extinctioncoefficient at 360 nm (˜5000 M⁻¹ cm⁻¹) increases the overall uncagingefficiency (72,75). The methoxymethylene group is preferred to protectthe 2- and 3-hydroxyls for two reasons: firstly, it is relatively smalland should not interfere with the binding to the IP₃ R; secondly, thisgroup is a mixed ortlioformate ester, which makes it very labile to acidhydrolysis and removable with acetic acid (86). An advantage of leavingthe methoxymethylene on the 2,3-hydroxyls is that phosphate migration iscompletely prevented and the absence of hydrogen-bond donors aidsmembrane permeation. In addition, the final active product cannot bemetabolized by the IP₃ -3-kinase. However, if one wished to deliverauthentic IP₃ that can be converted to 1,3,4,5-IP₄ in vivo, the3-hydroxyl should be unmasked by acetic acid before administering tocells. The formyl group usually remains on the axial hydroxyl group(87). The structures of a preferred caged, membrane-permeant IP₃derivative (IP₃ -CPM) with and without the methoxymethylene is shown inFIG. 1, together with their expected mode of action

The detailed synthesis of IP₃ -CPM is set forth in FIGS. 12 and 13. Thesynthesis started from racemic diol III-27. This compound was purifiedby recrystallization from the mixture of three regio-isomors ofmyo-inositol bisacetoridle. The commercial availability of this compoundprovides a route to prepare tho intermediate, triol V-8 (see FIGS. 12and 13). In order to cover the 6-hydroxyl with DMNB group, aregioselective alkylation method is needed. A few procedures have beenpublished for selective benzylation of the 6-hydroxyl in the presence ofthe 1 hydroxyl. These include Garegg's phase-transfer reaction (BnBr,Bu₄ NHSO₄, CH₂ CI₂, 5% NaOH, reflux)(93) and Fraser-Reid's tin chemistry(Bu₂ SnO, SnBr, toluene, reflux) (88,89). Similar selectivity isachievable with DMNB bromide due to its structural similarity to benzylbromide. Since nitrobenzyl groups are not very stable under stronglybasic conditions (90), Fraser-Reid's method is preferred because thisreaction is carried out under neutral conditions and gives betterselectivity. Thus, refluxing III-27 with dibutyl tinoxide gave acyclized dibutylstannylene intermediate. The reaction was carried out ina Dean-Stark apparatus to remove water. Direct addition of DMNB bromideand cesium fluoride to the dibutylstannylene intermediate affordedmonobenzylated product V-2 (FIG. 12). A very small amount of1-benzylated isomer was also formed, but it could be removed by flashchromatography.

In order to resolve the racemic myo-inositol derivatives, V-2 wasesterified with S-(-)-camphanic acid chloride to form a pair ofdiastereomers. S-(-)-camphanic acid chloride has been used before toresolve the racemic 4-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol(91), which was structurally similar to V-2. After these twodiastereomers are isolated as a mixture on a silica gel. 60 column, oneof the isomers (V-3) can be selectively recrystallized out of themixture. For comparison, another diastereomer, very close to V-3 on thinlayer chromatography (TLC), was purified to homogeneity in smallquantity by flash chromatography. The ¹ H-NMR spectra of these twocompounds are very similar except that the chemical shift of one of theinositol ring-protons is different.

The trans-acetonide of V-3 was selectively removed in the presence ofmore stable cis-acetonide. Esterification of V4 and deprotection ofcis-acetonide gave diol V-6. "Locking" the cis-diol of V-6 withmethoxymethylene group turned out to be nontrival. Even with a largeexcess of trimethyl orthoformate and strong catalysts, such as p-TsOH,TMS triflate or boron trifluoride diethyl etherate, no product (V-7) wasisolated. This problem appears to originate from the presence of the1-O-camphanate, which might be too bulky and block the 2- and3-hydroxyls. Accordingly, it is preferred that the camphanate of V-3 bereplaced with benzoate and the diol V-13 prepared following the samesynthetic route. This procedure is set forth in FIG. 13. Brief treatmentof V-14 with acetic acid hydrolyzed methoxymethylene group and formed aformate on the axial 2-hydroxyl group, as shown by ¹ H-NMR. Methanolysisof V-14 gave the intermediate, triol V-8. Standard phosphitylation,oxidation and removal of β-cyanoethyl groups provided trisphosphate V-16in 3 further steps.

Details of the synthesis are as follows:

Rac-2,3:4,5-di-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol(V-2): Diol III-37 (2 g, 7.9 mmol) and dibutyltin oxide (2 g, 7.9 mmol)were refluxed in 50 mL anhydrous toluene in a Dean-Stark apparatus. Thewater formed during the reaction was removed from the organic phase. 3 hlater, toluene was removed under vacuum. The residue was redissolved in30 mL DMF. CsF (1.5 9, 10 mmol) and 4,5-dimethoxy-2-nitrobenzylbromide(DMNB bromide, 3 g, 10.8 mmol) were then added and the mixturewas-stirred vigorously at room temperature overnight. The mixture wasdiluted with 40 mL CH₂ CI₂ and filtered through a glass fiber filter.After removal of the solvent under vacuum, the residue was purified on asilica gel 60 column (hexane/acetono). 2.25 g of product was obtained asa pale yellow solid (63%). ¹ H-NMR (CDCI₃): δ 1.42-1.67(m, 12H, CH₃),3.65(m, 1H), 3.9(dd, J=7.9, 1.8 Hz, 1H), 3.98(s, 3H, OCH₃), 4.05(s, 3H,OCH₃), 4.1 (m, 1H), 4.2(m, 1H), 4.4(t, J=7.2 Hz, 1H), 4.5(m, 1H), 5.1(d, J=2.8 Hz, 2H, CH2), 7.38(s, 1H), 7.76(s, 1H).

D-2,3;4,5-di-O-isopropylidene-6-O-(4,5-dimothoxy-2-nitrobenzyl)-myo-nositol1-(S)camphanate (V-3): Compound V-2 (3.07 g, 6.75 mmol) was dissolved in70 mL CH₂ CI₂. Triethylamine (2.4 mL, 17 mmol) and a catalytic amount of4-dimethylaminopyridine (DMAP, 50 mg) were included. S-(-)-camphanicacid chloride (1.7 g, 7.8 mmol) was added in one portion at 0° C. Afterstirring at room temperature for 3h, the solvent was removed and theresidue was purified on a silica gel 60 column (Et₂ O/CH₂ CI₂). 4.28 gof product (100%) which contained both diastereomers was obtained. Thiswas further separated by recrystallization from acetone/hexane (200 mL,˜1:1). 1.35 g of crystalline material was obtained from the first crop.The second crop gave another 0.40 g crystal which was the samediastersomer as the first crop as judged from ¹ H-NMR. This was laterconfirmed to be D-isomer by IP₂ binding assay. ¹ H-NMR (CDCI₃) δ0.9-2.05 (m, 12H), 2.45(m, 1H), 3.58(t, J=9 Hz, 1H), 3.84-4.02(m, 7H),4.37(dd, J=8.2. 6 Hz. 1H), 4.63(t, J=4.6 Hz, 1H), 5.1-5.3 (m, 4H),7.35(s, 1H), 7.7(s, 1H). Aliquots of mother liquor was purified again onflash chromatography (silica gel 60, E(2O/CH₂ CI₂) to obtain a smallsample of L-diastereomer for comparison.L-2,3:4,5-di-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol1-camphanate was a little bit more polar than its D-diastereomer on TLC.¹ H-NMR (CDCI₃): δ 0.9-2.05(m, 12H), 2.45(m, 1H), 3.58(t, J=9 Hz, 1H),3.84-4.02(m, 7H), 4.37(dd, J=8.2, 6Hz, 1H),4.58(t, J=4.6 Hz, 1H),5.1-5.3(m, 4H), 7.29(s, 1H), 7.7(s, 1H).

D-2,3:4,5-di-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol(V-9): Compound V-3 (1.81 g, 2.85 mmol) was saponified overnight in 25mL CH₂ CI₂ and 5 mL MeOH containing 1.5 g of K₂ CO₃. The mixture wasfiltered through a glass fiber filter and concentrated. It was thenpurified on a silica gel 60 column and used directly for the next step.

D-2,3:4,5-di-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-1-O-benzoyl-myo-inositol(V-1 0): The above compound and a small amount of DMAP (50 mg) wasdissolved in 15 mL pyridine. The mixture was stirred at room temperaturefor 1h and the solvent was removed under vacuum. The residue waspartitioned between CH₂ CI₂ and NH₄ CI solution. The organic layer wasdried over Na₂ SO₄ and concentrated. It was purified on a silica gel 60column and used directly for the next step.

D-2,3-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-1-O-benzoyl-myo-inositol(V-11): The above compound V-10 was dissolved in 10 mL CH₂ CI₂containing 2-mercaptoethanol(0.39 mL, 5.6 mmol). Boron trifluoridediethyl etherate (0.13 mL, 0.5 mmol) was then added. The reaction wasmonitored by TLC. In about half an hour the mixture was purified on asilica gel 60 column (CH₂ CI₂ /MeOH). ¹ H-NMR (CDCI₃): δ 1.35(s, CH₃),1.55(s, CH₃), 3.65(t, 1H), 3.85-4.2(m, 9H), 4.55(t, 1H), 5.1 (d, 1H),5.35(d, 1H), 5.47(dd, 1H), 7.1-7.6(m, 5H), 7.95(m, 2H).

D-2,3-O-isopropylidene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-1,4,5-tri-O-benzoyl-myo-inositol(V-12): Diol V-11 (0.65 g, 1.26 mmol) was dissolved in 4 mL pyridinecontaining a catalytic amount of DMAP (30 mg). Benzoyl chloride (0.44mL, 3,78 mmol) was added in portion and the reaction was continued atroom temperature for 2 h. After removal of the solvent under vacuum, theresidue was partitioned between CH₂ CI₂ and NH₄ CI solution. The organiclayer was dried over Na₂ SO₄ and concentrated. It was purified on asilica gel 60 column and 0.8 g of pale yellow syrup (94%) was obtained.This material was used directly for the next step.

D-6-O-(4,5-dimethoxy-2-nitrobenzyl)-1,4,5-tri-O-benzoyl-myo-inositol(V-13): Compound V-12 (0.8 g, 1.1 mmol) was dissolved in 10 mL CH₂ CI₂containing 2-mercaptoethanol(0.31 mL, 4.4 mmol). Boron trifluoridediethyl etherate (0.05 mL, 0.19 mmol) was then added. The reaction wasmonitored by TLC. In 3h, the reaction was quenched by addingtriethylamine (60 μL). The product was purified on a silica gel 60column (toluene/ethyl acetate) and 0.6 g of light yellow solid (80%) wasyielded. ¹ H-NMR (CDCI₃): δ 3.7(s, 3H, OCH₃), 3.85(s, 3H, OCH₃), 4.0(dd,J=9.5, 2.7 Hz, 1H, H₃), 4.35(t, J=2.6 Hz, 1H, H2), 4.58(t, J=10 Hz, 1H,H₆), 5.0-5.25(m, 2H), 5.3(dd, J=10.1, 2.6 Hz, 1H, H₁), 5.6-5.85(m, 2H,H₄ /H₅), 7.1-8.15(m, 17H).

D-2,3-O-methoxymethylene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-1,4,5-tri-O-benzoyl-myo-inositol(V-14): Diol V-13(0.6 g, 0.88 mmol) was mixed with 2 mL CH₂ CI₂ and 1 mLDMF. Boron trifluoride diethyl etherate (0.03 mL, 0.114 mmol) and excesstrimethyl orthoformate (1 mL) were added. After 5 h at room temperature,the reaction was quenched by adding triethylamine (60 μL). The solventwas removed under vacuum and the residue was purified on a silica gel 60column (hexane/ethyl acetate). 0.5 g of light yellow syrup (78%) wasyielded and was used directly for the next step.

D-2,3-O-methoxymethylene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol(V-8): V-14 (0.5 g, 0.69 mmol) was dissolved in 2 mL CH₂ CI₂ and 12 mLMeOH. K₂ CO₃ (1.4 g, 10.3 mmol) was added, and the reaction wascontinued at room temperature for 24 h. The mixture was filtered througha glass fiber filter and concentrated. The residue was first passedthrough a short silica gel 60 column to remove salt. It was thenpurified on a silica gel 60 column (ethyl acetate/MeOH). A small amountof incompletely saponified products were eluted out before the desiredproduct. One of them was dibenzoylated product, MS m/z 594.4(M-OCH₃)⁺,expected 594.56 (C₃₁ H₃₁ O₁₃ N₁ -OCH₃)⁺, and the other wasmonobenzoylated product, MS m/z 490.3(M-OCH₃)⁺, expected 490.45 (C₂₄ H₂₇O₁₂ N₁ -OCH₃)⁺. 150 mg of product was obtained as a pale yellow solidafter dried in vacuum. ¹ H-NMR (CDCI₃ /d-MeOH, 10:1): δ 3.37(s, 3H,OCH₃),3.4-4.1 (m, 11H), 5.2(s, 3H), 5.37(t, 1H), 7.45(d, 1H), 7.52(s,1H).MS m/z 386.1 (M-OCH₃)⁺, expected 386.34(C₁₇ H₂₃ O₁₁ N₁ -OCH₃)⁺.

D-2,3-O-methoxymethylone-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol1,4,5-trisphosphate hexakis(β-cyanoethyl)ester (V-15): To a solution of75 mg (0.18 mmol) of triol V-8 and 0.24 g (0.9 mmol) ofN,N-diisopropyl-bis(β-cyanoethyl)phosphoramidite in 1 mL CH₃ CN wasadded 49 mg (0.7 mmol) of tetrazole dissolved in 1 mL CH₃ CN. Afterstirring at room temperature for 0.5 h, 0.4 mL tert-butyl hydroperoxide(3M solution in 2,2,4-trimethylpentane) in 1 mL CH₂ CI₂ was added in oneportion at 0° C. After addition, the solution was warmed up to roomtemperature in 0.5 h and another 0.1 mL t-BuOOH solution was added. 10min later, the solvent was removed under vacuum. The residue was loadeddirectly onto a silica gel 60 column and eluted with CH₂ CI₂ /MeOH(15:1). Evaporation of solvent provided 0.14 g (79% for 2 steps) ofproduct as a light yellow glass. MS m/z 944.39(M-OCH₃)⁺, expected944.69(C₃₅ H₄₄ O₂₀ N₇ P₃ -OCH₃)⁺.

D-2,3-O-methoxymethylene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol1,4,5-trisphosphate (V-16), ammonium salt: The above trisphosphate V-15(0.14 g, 0.143 mmol) was suspended in 1 mL methanol and 6 mLconcentrated NH₄ OH. The solution was heated at 70° C. for 1.5 h andlyophilized. The resulting pale yellow solid was used directly for thenext step.

D-2,3-O-methoxymethylene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-Inositol1,4,5-trisphosphate hexakis(propionyloxymethyl)ester (V-1): Half of theabove material was mixed vigorously with 0.5 mL CH₃ CN and 0.1 mL DIEA.The mixture was then dried under vacuum. This procedure was repeated atleast three times until a homogenous solution was obtained after addingCH₃ CN/DIEA (Sonication may be used to help solubilization). At thistime, the counter-ion of the phosphate had presumably been exchangedfrom ammonium ion to diisopropylethylammonium ion. After a final roundof drying, the pale yellow solid was suspended in 0.5 mL CH₃ CN and 0.1mL DIEA. 100 mg (0.55 mmol) of bromomethyl propionate was added to thissolution. After stirring for 1 day, another 40 mg of bromomethylpropionate was added and the reaction was continued for another 24 h.The solvent and excess reagent were evaporated under vacuum. Theremaining mixture was purified on silica gel column using ethyl acetateas eluant. 10 mg of product (12% for 2 steps) was obtained as a lightyellow glass. ³¹ P-NMR (CDCI₃): δ (-4.6, broad) MS: exact mass calcd for(C₄₁ H₆₂ O₃₂ NP₃ +CS⁺) 1305.1444, observed 1305.1481 (2.8 ppm).

D-2,3-O-methoxymethylene-6-O-(4,5-dimethoxy-2-nitrobenzyl)-myo-inositol1,4,5-trisphosphate hexakis(acetoxymethyl)ester: This was synthesized ina similar manner as above except using bromomethyl acetate asesterification reagent. 8 mg product (10%) as a light yellow glass. ³¹P-NMR (CDCI₃): δ-4.55(s, 1P), -4.65(s, 2P). MS: exact mass calcd for(C₃₅ H₅₀ O₃₂ NP₃ +Cs⁺) 1222.0583, observed 1222.0533 (4.1 ppm).

Exemplary Syntheses of Analogs of IP₃ -CPM

To produce the compounds with R₃ being photolabile protecting groupsother than 4,5-dimethoxy-2-nitrobenzyl as described above, the4,5-dimethoxy-2-nitrobenzyl bromide presently used to convert III-37 toV-2 (see FIG. 12) is replaced by the chloride, bromide, iodide, ortosylate of the appropriate photolabile protecting group, i.e. (R₃)CI,(R₃ (Br, (R₃)I, or (R₃)OTs.

To produce the compounds with R₁ and R₂ together being --CH₂ --,--CHMe--, --CMe₂ --, --CMe(OMe)--, or --C(OMe)₂ --, the trimethylorthoformate used in the preparation of V-14 (see FIG. 13) is replacedrespectively by dimethoxymethane, acetaldehyde dimethyl acetal2,2-dimethoxypropane, trimethyl orthoacetate, or tetramethylorthocarbonate, as known in standard methods for preparing cyclic ketalsand orthoesters.

To produce the structures R₂ =H and R₁ =H or --CHO, the synthetic schemeoutlined in FIG. 16 can be followed. Brief incubation of V-1 with anacetic/trifluoroacetic acid mixture removes the methoxymethylenebridging the 2,3-hydroxyls and gives the above products, both of whichhave free 3-hydroxyls (R₂ =H). Longer incubations will shift the productdistribution towards de-esterifieation of the 2-hydroxyl (R₁ =H). Thesame synthetic strategy is applicable to other acid labile groups, suchas acetonide, orthoacetate, or orthocarbonate. For example, thestructure with R₁ =--COCH₃ would result from brief acid treatment of theorthoacetate, in which R₁ and R₂ together had been --CMe(OMe)--. Thestructure with R₁ =--COOCH₃ would result from brief acid treatment ofthe orthocarbonate, in which R₁ and R₂ together had been --C(OMe)₂ --.

An exemplary synthesis procedure for making analogs where X=S, is setforth in FIG. 17. The tert-butyl hydroperoxide used in the synthesis ofV-15 would be replaced by a sulfur donor such as benzoyl disulfide. Forexample, the caged membrane-permeant derivative of myo-inositol1,4,5-trisphosphorothioate (IPS₃ -CPM) would be synthesized by a slightmodification of the synthetic strategy used for making IP₃ -CPM.Phosphitylation of triol V-8 gives a phosphite triester intermediatewhich provides the tris(phosphorothioate) upon treatment with benzoyldisulfide. Removal of β-cyanoethyl groups and esterification of theresulting tris(phosphorothioate) will give the target molecule with X=S.If a free 3-hydroxyl group is desired, the orthoformate or other acidlabile moieties (acetonide, orthoacetate, or orthocarbonate) bridgingthe 2- and 3-positions can be similarly removed by acid treatment asdescribed above.

An exemplary synthesis procedure for making analogs where R₂=--P(O)(OR₄)(XR₄), i.e. ##STR17## such as caged membrane-permeantmyo-inositol 1,3,4,5-tetrakisphosphate (IP₄ -CMP), is set forth in FIG.18. The 3-hydroxyl of V-8 would be deprotected by acid (e.g. acetic acidwith a catalytic amount of trifluoroacetic acid), leaving a formateester on the 2-position. Standard phosphitylation, oxidation and removalof β-cyanoethyl groups analogous to the preparation of V-15 will installphosphates on the 1,3,4,5-positions. The 2-hydroxyl will bede-esterified in the process of cleaving the β-cyanoethyl groups withammonia. Esterification of the tetrakisphosphates with bromomethylacetate, propionate or butyrate yields the target molecules in which R₁=H. If R₁ =--CHO, --COCH₃, or --COOCH₃ is desired, the 2-hydroxyl can bere-esterified at this stage using acetic formic anhydride, aceticanhydride, or methyl chloroformate respectively, since the 2-positioncarries the only free hydroxyl. It should be noted that step d in FIG.17 and step 3 in FIG. 18 are optional and are needed only if one wishesR₂ to be H, i.e. for 3-hydroxyl to be free.

Bovine cerebellum microsomal fractions, which are a rich source of IP₃binding protein, were prepared and used for ³ H!IP₃ binding assay. Thepreparation bound to ³ H!IP₃ with high affinity and the binding wascompetitively inhibited by cold D-IP₃ in a dose dependent manner. TheIC₅₀ of D-IP₃ was about 8 nM. In agreement with the published data,L-IP₃ had negligible affinity to IP₃ R. Thus, if V-16 was derived fromD-isomer, its photolysed product, i.e.D-O-2,3-methoxymethylene-myo-inositol 1,4,5-trisphosphate, should have ahigh affinity to the IP₃ R; otherwise its photolysed product would haveminimum effect on the binding of ³ H!IP₃ to the receptor.

The caged IP₃ derivative V-16 showed negligible binding to cerebellarmicrosomes because its 6-hydroxyl was blocked. Upon photolysis at 366nm, V-16 displayed a UV-dose-dependent increase of binding to thereceptor. In about 2 hours, the binding leveled off because longerexposure to UV light did not change the binding activity significantly,indicating most of V-16 was photolyzed. At this point, the crudephotolysis product from 100 pmol V-16 was equivalent in binding activityto 32 pmol of authentic IP₃. Assuming the chemical yield of the uncagedproduct is 100%, its affinity for the IP₃ receptor is only 3-fold weakerthan that of real IP₃. If the chemical yield were lower than 100%, theaffinity of the photolysis product would have to be even closer to thatof IP₃. This example shows that the photolysis product of V-16 isD-2,3-methoxymethylene-myo-inositol 1,4,5-trisphosphate, which meansthat V-16 was derived from precursors of D-configuration. The absolutestereochemistry of V-3 and its diastereomer was thus assigned.

In Vivo Tests of Caged Membrane-Permeant IP₃

In 1321N1 astrocytoma cells, extracellular addition of 20 μM IP₃ -CPMhad no effect on Ca²⁺ !_(i) even after incubation for over 5 minutes(FIG. 14(a)). The Ca²⁺ !_(i) was monitored using the calcium indicatorfluo-3 rather than fura-2 to keep the excitation wavelength of theindicator (490 nm) far from the uncaging wavelength (360 nm) of DMNBgroup. Subsequent illumination of the cells with UV (360 nm) through themicroscope objective for a short period of time (10 seconds) caused analmost saturating Ca²⁺ !_(i) increase. Compared to the photolyzingset-up (direct illumination by a hand mercury lamp) used for the IP₃-binding assay, the optical throughput from the source lamp to thespecimen is much higher in a microscope, so 10 seconds (as opposed to 2hours in IP₃ binding assay) of photolysis was sufficient to produceenough IP₃ to elicit Ca²⁺ !_(i) increase. The same amount of UV alonehad no effect at all on Ca²⁺ !_(i) in cells not treated with IP₃ -CPM.After recovering from uncaging of IP₃ -CPM, the cells responded tosubsequent carbachol and thapsigargin stimulation normally. Ionomycinfurther raised Ca²⁺ !_(i) to a saturating level.

A similar response to IP₃ -CPM was also observed when the experiment wasdone in calcium free medium (FIG. 14(b)). This confirmed that the Ca²⁺!_(i) increase due to uncaging of IP₃ -CPM resulted from internal Ca²⁺release, which was consistent with the normal mechanism of IP₃ action.Carbachol and thapsigargin were still able to cause further Ca²⁺release, suggesting that the internal stores were only partially emptiedby the first dose of uncaged IP₃ analog, or that they had substantiallyrefilled with Ca²⁺ after that agonist had been metabolized. Ionomycinwas added at the end of the experiment, but it only caused a small andtransient Ca²⁺ !_(i) increase, possibly from some IP₃ -insensitivestores.

Another example was carried out in P388D₁ macrophage-like cells. Theresults were the same as the astrocytoma cells and are shown in FIG. 15.

Extracellular addition of 20 μM IP₃ -CPM had no effect on Ca²⁺ !_(i)even after incubation for over 5 minutes. Photolysis for 10 seconds (360nm) released a fair amount of Ca²⁺ from internal stores. Compared to theexperiments done in astrocytes, Ca²⁺ influx in P388D₁ cells afteruncaging was less obvious, and subsequent stimulation with PAF gavesmaller Ca²⁺ !_(i) increase, possibly the internal stores were notrefilled yet. Studies by Asmis et al. (92) have shown that, in P388D₁macrophage-like cells, PAF stimulated PGE₂ production is mediatedthrough two separate signals: IP₃ induced Ca²⁺ !_(i) increase andanother unidentified signal. IP₃ -CPM might serve as a new tool fordetailed study of PGE₂ formation and AA release in relation to IP₃ andCa²⁺ !_(i) in these cells. One of the major advantages of using permeantIP₃ derivatives is that experiments can be easily carried out in apopulation of cells, which is required for the measurement of AA releaseand PGE₂ formation.

The above examples show that IP₃ -CPM crossed the cell membrane and itsPM esters underwent intracellular hydrolysis. The resulting caged IP₃analog (V-16) was inactive in releasing Ca²⁺, which was consistent withthe in vitro ³ H!IP₃ binding experiment. Upon photolysis, the caged IP₃analog V-16 rapidly formed active D-2,3-methoxymethylene-myo-inositol1,4,5-trisphosphate, which has an affinity to IP₃ R at least one thirdthat of IP₃, and induced internal Ca²⁺ release. Furthermore, V-16, onceit was released intracellularly, was well retained in cells and was alsometabolically fairly stable.

In human embryonic kidney cells (HEK cells), the extracellular IP₃ -CPMwas washed away after the cells had been loaded with 50 μM of IP₃ -CPMfor 20 minutes. Photolysis of the cells in the field for 1 ms with acapacitor-discharge flash lamp resulted in a transient increase in Ca²⁺!_(i). The ability of the cell's Ca²⁺ !_(i) to respond to each flashpersisted for up to a dozen repetitions. Cells in the same dish whichhad not been exposed to UV light were kept in the dark for over 6 hours.Photolysis of these cells still gave Ca²⁺ !_(i) increases similar tothose obtained immediately after being loaded with IP₃ -CPM. This showsthat caged IP₃ (V-16) was resistant to phosphatase degradation and couldbe trapped inside cells for a long period of time without losing much ofits activity. The photolysis product of V-16 can not be phosphorylatedinto 1,3,4,5-IP₄. However, pretreatment of IP₃ -CPM with acetic acidwill unmask the 3-hydroxyl and enable the intracellular phosphorylationof delivered IP₃.

Another caged, membrane permeant IP₃ derivative, 1,4,5-triphosphatehexakis (acetoxymethyl) ester (IP₃ -CAM), was also synthesized from V-16using AM bromide as esterification reagent. Biological tests of thiscompound in astrocytes gave similar response as IP₃ -CPM, yet higherconcentration of IP₃ -CAM (30 μM) and longer incubation time (more than15 minutes) were required, possibly because of the decreased membranepermeability of the prodrug.

As is apparent from the above examples, a number of caged andmembrane-permeant IP₃ derivatives can be prepared using the abovedescribed general and specific procedures. These cage derivativesdisplay a faster onset of activity which can be triggered by a flash ofUV light and can be controlled with millisecond temporal resolution. In1321N1 astrocytoma cells, extracellular concentrations of only 10⁻⁶ to10⁻⁵ M of IP₃ -CPM were able to elicit maximal Ca²⁺ release uponuncaging. In addition, IP₃ -CPM has all the hydroxyls of IP₃ blocked bysuitable protecting groups, thus eliminating the possibility ofphosphate randomization during the delivery process. This improves theefficiency and isomeric specificity of IP₃ delivery. The hydrolysisproduct of IP3-CPM, i.e. caged IP₃ (V-16), was fairly resistant tophosphatase degradation. UV exposure of the HEK cells loaded with IP₃-CPM 6 hours previously still gave full-scale Ca²⁺ !_(i) increases.These properties are useful for various biological applications. Inaddition, IP₃ -CPM was found to be chemically more stable thancommercially available caged IP₃ where one of the vicinal 4- or5-phosphates is randomly masked by one NPE group.

Permeant inositol polyphosphate derivatives as described above areuseful to investigate longer-term effects on cells not mediated throughcytosolic Ca²⁺ !_(i). For example, possible effects on proteinsynthesis, phosphorylation, proliferation (49), or gene expression arerelatively difficult to study on microinjected, patch-clamped, orpermeabilized cells.

Inositol polyphosphates are among the most ubiquitous and importantintracellular second messengers. They are essential for hormonesecretion and action, smooth muscle contraction, immune activation,fertilization, and neuronal function. As previously mentioned, the bestknown inositol polyphosphate is myo-inositol-1,4,5-triphosphate (IP₃),which acts by releasing Ca²⁺ from intracellular stores. IP₃ undergoesfurther metabolism to a series of inositol polyphosphates of largelyunknown biochemical function. A major problem in investigating thebiological functions of inositol polyphosphates is their membraneimpermeability. All existing techniques for delivering exogenousinositol polyphosphates into cells require puncture of their plasmamembranes by techniques that jeopardize the cells' viability and areoften applicable only to single cells. The inositol polyphosphatederivatives in accordance with the present invention provide a solutionof this problem because they are membrane-permeant and they regenerateinto biologically active inositol polyphosphates once inside the cell.

The acyloxyalkyl esters of phosphate-containing second messengers inaccordance with the present invention may be used in any situation whereit is desirable to introduce the second messenger into a cell withoutpuncturing the cell membrane or otherwise adversely affecting the cell.The present invention is useful as a replacement for the existingmethods which rely on microinjection, patch-clamp and electroporationtechniques to introduce phosphate-containing second messengers intocells.

The acyloxyalkyl esters are introduced into the selected cells by simplyexposing the cells to the esters-in cell growth media or other suitablesolution. The amount of ester which is introduced into the cell can becontrolled by reducing or increasing the concentration of ester presentin the cell growth media. The amount of ester which permeates into thecell may also be controlled by limiting the amount of time that thecells are left exposed to the ester.

When using caged compounds, the additional step of exposing the cells toUV radiation is required. UV light at wavelengths around 360 nm ispreferred. However, other wavelengths within the UV spectrum may be usedprovided that the selected wavelength is sufficient to uncage theprotected compounds. The particular wavelength and degree of exposurerequired to uncage the protected compounds can be established for eachcase using well known procedures for exposing cells to UV radiation.

Having thus described exemplary embodiments of the present invention, itwill be understood by those skilled in the art that the withindisclosures are exemplary only and that the invention is only limited bythe following claims.

BIBLIOGRAPHY

1. Hardie, D. G.(1991) Biochemical Messengers: Hormones,Neurotransmitters and Growth Factors, Champman & Hall, London.

2. Robinson, G. A., Butcher, R. W., and Sutherland, E. W. (1971) CyclicAMP, Academic Press, New York.

3. Corbin, J. D., Johnson, R. A., eds (1988) Methods in Enzymology:Initiation and Termination of Cyclic Nucleotide Action, Academic Press,Inc., San Diego.

4. Goy, M. F.(1991) Trends Neurosci. 14, 293-299.

5. Berridge, M. J. and Irvine, R. F. (1989) Nature 341, 197-205.

6. Meyer, R. B., Jr. (1980) in Burger's Medicinal Chemistry (Wolff, M.E., ed.) pp. 1201-1224, Wiley, New York.

7. Polokoff, M. A., Bencen, G. H., Vacca, J. P., deSolms, S. J., Young,S. D., and Huff, J. R. (1988) J. Biol. Chem. 263, 11922-11927.

8. Henion, W. F., Sutherland, E. W., and Posternak, T. (1967) Biochim.Biophys. Acta 148, 106-113.

9. Roche, E. B., ed. (1987) Bioreversible Carriers in Drug DesignPergamon Press, New York.

10. Falbriard, J.-G., Posternak, T., and Sutherland, E. W. (1967)Biochim. Biophys. Acta 148, 99-105.

11. Jansen, A. B. A. and Russell, T. J. (1965) J. Chem. Soc. 2127-2132.

12. Tsien, R. Y.(1981) Nature 290, 527-528.

13. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem.260, 3440-3450.

14. Tsien, R. Y.(1989) Methods Cell Biol. 30, 127-156.

15. Srivastva, D. N. and Farquhar, D. (1984) Bioorg. Chem. 12, 118-129.

16. Iyer, R. P., Phillips, L. R., Biddle, J. A., Thakker, D. R., Egan,W., Aoki, S., and Mitsuga, H. (1989) Tetrahedron Lett. 30, 7141-7144.

17. Sastry, J. K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W.,Arlinghaus, R. B., and Farquhar, D. (1992) Mol. Pharmacol. 41, 441-445.

18. Freed, J. J., Farquhar, D., and Hompton, A. (1989) Biochem.Pharmacol. 38, 3193-3198.

19. Saperstein, R., Vicario, P. P., Strout, H. V., Brady, E., Slater, E.E., Greenlee, W. J., Ondeyka, D. L., Patchett, A. A., and Hangauer, D.G. (1989) Biochemistry 28, 5694-5701.

20. Walker, J. W., Reid, G. P., McCray, J. A., and Trentham, D. R.(1988) J. Am. Chem. Soc. 110, 7170-7177.

21. Nerbonne, J. M., Richard, S., Nargeot, J., and Lester, H. A. (1984)Nature 310, 74-76.

22. Engels, J. and Schlaeger, E. -J. (1977) J. Med. Chem. 20, 907-911.

23. Walker, J. W., Feeney, J., and Trentham, D. R. (1989) Biochemistry28, 3272-3280.

24. Gurney, A. M. and Lester, H. A. (1987) Physiol. Rev. 67, 583-617.

25. McCray, J. A. and Trentham, D. R. (1989) Annu. Rev. Biophys.Biophys. Chem. 18, 239-270.

26. Berridge, M. J. and Irvine, R. F. (1989) Nature 341, 197-205.

27. Irvine, R. F. and Moor, R. M. (1986) Biochem. J. 240, 917-920.

28. Irvine, R. F. (1990) FEBS Letters 263, 5-9.

29. Morris, A. P., Gallacher, D. V., Irvine, R. F., and Petersen, O. H.(1987) Nature 330, 653-655.

30. Changya, L., Gallacher, D. V., Irvine, R. F., Potter, B. V. L., andPetersen, O. H. (1989) J. Membrane Biol. 109, 85-93.

31. Boynton, A. L., Dean, N. M., and Hill, T. D. (1990) Biochem.Pharmacol. 40, 1933-1939.

32. Hill, T. D., Dean, N. M., and Boynton, A. L. (1988) Science 242,1176-1178.

33. Crossley, I., Swann, K., Chambers, E., and Whitaker, M. (1988)Biochem. J. 252, 257-262.

34. Snyder, P. M., Krause, K.-H., and Welsh, M. J. (1988) J. Biol. Chem.263, 11048-11051.

35. Tsien, R. W. and Tsien, R. Y. (1990) Annu. Rev. Cell Biol. 6,715-760.

36. Bird, G. St J., Rossier, M. F., Hughes, A. R., Shears, S. B.,Armstrong, D. L., and Putney, Jr., J. W. (1991) Nature 352, 162-165.

37. Balla, T., Sim, S. S., Iida, T., Choi, K. Y., Catt, K. J., and Rhee,S. G. (1991) J. Biol. Chem. 266, 24719-24726.

38. Grynkiewicz, G. and Tsien, R. Y. (1987) Pol. J. Chem. 61, 443-447.

39. Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S.,and Tsien, R. Y. (1991) Nature 349, 694-697.

40. Dharmsathaporn, K., Mandel, K. G., Masui, H., and McRoberts, J. A.(1985) J. Clin. Invest. 75, 462-470.

41. Madara, J. and Dharmsathaporn, K. (1985) J. Cell Biol. 101,2124-2133.

42. Dharmsathaporn, K., Mandel, K. G., McRoberts, J. A., Tisdale, L. D.,and Masui, H. (1984) Am. J. Physiol. 264, G204-G208.

43. McRoberts, J. A. and Barrett, K. E. (1989) Modern Cell Biology(Mathi, K. S. and Valeulich, J. D., eds) pp. 235-265, Alan R. Liss,Inc., New York.

44. Sammak, P. J., Adams, S. R., Harootunian, A. T., Schliwa, M., andTsien, R. Y. (1992) J. Cell Biol. 117, 57-72.

45. Taylor, S. S., Buechler, J. A., and Yonemoto, W. (1990) Annu. Rev.Biochem. 59, 971-1005.

46. Barrett, K. E. and Dharmsathaporn, K. (1991) Textbook ofGastroenterology (Yamada, T., ed) pp. 265-294, J. B. Lippincott Co.,Philadelphia.

47. Schliwa, M. (1975) Microtubules and Microtubule Inhibitors (Borgers,M. and de Brabender, M., eds) pp. 215-228, Elsevier Science, Amsterdam.

48. Beebe, S. J., Blackmore, P. F., Chrisman, T. D., and Corbin, J. D.(1988) Methods Enzymol. 159, 118-139.

49. Smirnova, L. I., Malenkovskaya, N. A, Preddoditelev, D. A., andNifant'ev, E. E. (1980) Zh. Org. Khim. 16, 1011-1019.

50. Angyal, S. J. and Tate, M. E. (1965) J. Chem. Soc. 6949-6955.

51. Tegge, W. (1986) Ph. D. Thesis University of Bremen-FRG.

52. Lee, H. W. and Kishi, Y. (1985) J. Org. Chem. 50, 4402-4404.

53. Billington, D. C., Baker, R., Kulagowski, J. J., Mawer, I. M.,Vacca, J. P., deSolms, S. J., and Hugg, J. R. (1989) J. Chem. Soc.Perkin Trans. 1 1423-1429.

54. Baudin, G., Glanzer, B. I., Swaminathan, K. S., and Vasella, A.(1988) Helv. Chim. Acta 71; 1367-1378.

55. Tegge, W. and Ballou, C. E. (1989) Proc. Natl. Acad. Sci. USA 86,94-98.

56. Perich, J. W. and Johns, R. B. (1987) Tetrahedron Lett. 28, 101-102.

57. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., andDawson, A. P. (1990) Proc. Natl. Acad. Sci. USA 87, No. 7, 2466-2470.

58. Tsien, R. Y. and Harootunian, A. T. (1990) Cell Calcium 11, 93-109.

59. Harootunian, A. T., Kao, J. P. Y., Paranjape, S., Adams, S. R.,Potter, B. V. L., and Tsien, R. Y. (1991) Cell Calcium 12, 153-164.

60. Harootunian, A. T., Kao, J. P. Y., Paranjape, S., and Tsien, R. Y.(1991) Science 251, 75-78.

61. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Scient 252,443-446.

62. Kuno, M. and Gardner, P. (1987) Nature 236, 301-304.

63. Penner, R., Matthews, G., and Neher, E. (1988) Nature 334, 499-504.

64. Schultz, C. et al. (1993) Journal of Biological Chemistry Vol. 268,No. 9, 6316-6322.

65. Oozaki, S. et al., Tetrahedron Lett. 27 (1986), 3157; Dreef, C. E.et al., Rec. Trav. Chim. Pays-Bas. 107 (1988), 395.

66. Billington, D. C. et al., J. Chem. Soc. Chem. Communications (1987),101 1; Baudin, G. et al., Helv. Chim. Acta 71 (1988) 1367.

67. Kozikowski, A. P. et al., J. Am. Chem. Soc. 115 (1993), 4429.

68. Kozikowski, A. P. et al., J. Am. Chem. Soc. 112 (1990), 7403.

69. Bannwarth, W. et al., Helv. Chim. Acta. 70 (1987), 175.

70. Euranto, E. K. et al., Acta. Chem. Scand. 20 (1966), 1273.

71. Randriamampita, C and Tsien, R. Y. (1993) Nature 364:809-814.

72. McCray, J. A. & Trentham, D. R. (1989) Annu. Rev. Biophys. Biophys.Chem. 18, 239-270.

73. Adams, S. R. & Tsien, R. Y. (1993) Annu. Rev. Physiol. 55, 755-784.

74. Walker, J. W., Reid, G. P. & Trentham, D. R. (1989) Methods Enzymol.172, 288-301.

75. Wootton, J. F. &Trentham, D. R. (1989) NATO ASI Ser. C 272

76. Lev-Ram, V., Makings, L. R., Keitz, P. F., Kao, J. P. & Tsien, R. Y.(1995) Neuron 15, 407-415.

77. Makings, L. R. & Tsien, R. Y. (1994) J. Biol. Chem. 269, 6282-6285.

78. Krafft, G. A., Sutton, W. R. & Cummings, R. T. (1988) J. Am. Chem.Soc. 110, 301.

79. Adams, S. R., Kao, J. P. Y. & Tsien, R. Y. (1988) J. Am. Chem. Soc.110, 3212.

80. Tsien, R. Y. & Zucker, R. S. (1986) Biophys. J. 50, 843-853.

81. Callaway, E. M. & Katz, L. C. (1993) Proc. Natl. Acad. Sci U.S.A.90, 7661-7665.

82. Wilcox, M., Viola, R. W., Johnson, K. W., Billington, A. P.,Carpenter, B. K., McCray, J. A., Guzikowski, A. P. & Hess, G. P. (1990)J. Org. Chem. 55, 1585.

83. Corrie, J. E., DeSantis, A., Katayama, Y., Khodakhah, K., Messenger,J. B., Ogden, D. C. & Trentham, D. R. (1993) J. Physiol. (Lond) 465,1-8.

84. Walker, J. W., Somlyo, A. V., Goldman, Y. E., Somlyo, A. P. &Trentham, D. R. (1987) Nature 327, 249-252.

85. Walker, J. W., Feeney, J. & Trentham, D. R. (1989) Biochemistry28,3272-3280.

86. Greene, T. W. & Wuts, P. G. M. (1991) Protective Groups in OrganicSynthesis, John 219 Wiley & Sons: New York

87. Hanessian, S. & Roy, R. (1985) Can. J. Chem. 63, 163.

88. Yu, K. & Fraser-Reid, B. (1988) Tetrahedron Lett. 26, 979.

89. Yu, K., Ko, K. & Fraser-Reid, B. (1988) Synthetic Comm. 18, 465.

90. Buncel, E. (1984) Electron Deficient Aromatic- andHeteroaromatic-Base Interactions, Elsevier: New York

91. Vacca, J. P., Jane deSolms, S., Huff, J. R., Billington, D. C.,Baker, R., Kulagowski, J. J. & Mawer, I. M. (1989) Tetrahedron 45, 5679

92. Asmis, R., Randriamampita, C., Tsien, R. Y. & Dennis, E. A. (1994)Biochem. J. 298, 543.

93. Garegg, P., Iverson, T., Johansson, R. & Lindberg, B. (1984)Carbohydr. Res. 130, 322.

What is claimed is:
 1. A compound having the formula ##STR18## whereinR₁ is H, --CHO, COOCH₃ or --COCH₃ ; R₂ is H or --P(O)(OR₄)(XR₄); or R₁and R₂ together are --CH₂ --, --CHMe--, --CMe₂ --, --CH(OMe)--,--CMe(OMe)-- or --C(OMe)₂ --; R₃ is a photolabile protecting groupselected from the group consisting of ##STR19## wherein R₆ =H, Me, CH₃CO; R₄ is ##STR20## X is O or S.
 2. A compound according to claim 1wherein X is O.
 3. A compound according to claim 2 wherein R₁ and R₂together are --CH(OMe).
 4. A compound according to claim 2 wherein R₃ is##STR21##
 5. A compound according to claim 2 wherein R₄ is ##STR22## 6.A compound according to claim 4 wherein R₄ is ##STR23##
 7. A compoundaccording to claim 3 wherein R4 is ##STR24##
 8. A compound according toclaim 3 wherein R₃ is ##STR25##
 9. A compound according to claim 8wherein R₄ is ##STR26##
 10. A method for introducingphosphate-containing second messengers into a cell without disruptingthe cell membrane, said method comprising the steps of:contacting saidcell with a compound having the formula ##STR27## wherein R₁ is H,--CHO, COOCH₃ or --COCH₃ ; R₂ is H or --P(O)(OR₄)(XR₄); or R₁ and R₂together are --CH₂ --, --CHMe--, --CMe₂ --, --CH(OMe)--, --CMe(OMe)-- or--C(OMe)₂ --; R₃ is a photolabile protecting group selected from thegroup consisting of ##STR28## wherein R₆ =H, Me, CH₃ CO; R₄ is ##STR29##; and X is O or S; said contact being for a sufficient time to providepermeation of said caged compound into said cell; and exposing said cellto a sufficient amount of ultraviolet radiation for a sufficient time touncage said caged compound.
 11. A method according to claim 10 wherein Xis O.
 12. A method according to claim 11 wherein R₄ is ##STR30##
 13. Amethod according to claim 11 wherein R₁ and R₂ together are --CH(OMe).14. A method according to claim 11 wherein R₃ is ##STR31##
 15. A methodaccording to claim 14 wherein R₄ is ##STR32##
 16. A compound accordingto claim 13 wherein R4 is ##STR33##
 17. A method according to claim 13wherein R₃ is ##STR34##
 18. A compound according to claim 17 wherein R₄is ##STR35##